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Conference Registration (Lower Atrium)

Welcome Reception (Huntley Dining Room) Gala Dinner (Lone Peak Pavilion)

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Lower Atrium Registration Table ~ Oral Presentation Drop-Off ~ Breaks

Jefferson Room Concurrent Scientific Sessions ~ ASR Business Meeting

Madison Room Poster Sessions

Gallatin Room General Sessions ~ Scientific Sessions

Huntley Dining Room All Meals *Sunday Evening’s Gala Dinner will be held in the Lone Peak Pavilion*

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2016 Schedule-at-a-Glance Saturday, June 11th 3-6:45 p.m. Conference Registration – Lower Atrium 6-8 p.m. Welcome Reception – Huntley Dining Room 7-8 p.m. Invited Historical Lecture – Huntley Dining Room

Sunday, June 12th 7:15-8:15 a.m. Breakfast Buffet – Huntley Dining Room Oral presentation drop-off – Lower Atrium 8:15-9 a.m. Howard Ricketts Memorial Lecture – Gallatin Room 9-9:40 a.m. Plenary Presentation – Gallatin Room 9:40-10 a.m. Coffee Break – Lower Atrium Oral presentation drop-off – Lower Atrium Poster Session 1 set-up – Madison Room 10 a.m.-12 p.m. Concurrent Sessions – Scientific Sessions 1: Epidemiology, Ecology, and Emerging Diseases – Gallatin Room 2: Coxiella/Bartonella Pathogenesis – Jefferson Room 12-1:20 p.m. Buffet Luncheon – Huntley Dining Room 1:30-2:45 p.m. Poster Session 1 and Refreshments – Madison Room 3-5 p.m. Scientific Session 3: Host-Pathogen Interactions l – Gallatin Room 5:15-6:15 p.m. ASR Business Meeting and Election of New Officers – Jefferson Room 7-9 p.m. Gala Dinner – Lone Peak Pavilion

Monday, June 13th 7-8 a.m. Breakfast Buffet – Huntley Dining Room Oral presentation drop – Lower Atrium Poster Session 2 set-up – Madison Room 8-10 a.m. Concurrent Sessions – Scientific Sessions 4: Vector Biology & Vector-Pathogen Interactions – Gallatin Room 5: Infection, Diagnosis, and Treatment – Jefferson Room 10 a.m.-12 p.m. Poster Session 2 & Refreshments – Madison Room Noon & Beyond Free afternoon to explore!

Tuesday, June 14th 7-8 a.m. Breakfast Buffet – Huntley Dining Room Oral presentation drop-off – Lower Atrium 8-10 a.m. Concurrent Sessions – Scientific Sessions 6: Host-Vector Pathogen Interactions ll – Gallatin Room 7: Innate and Adaptive Immunity – Jefferson Room 10-10:30 a.m. Refreshment Break – Lower Atrium Oral presentation drop-off – Lower Atrium 10:30 a.m.- Scientific Session 8: Genetics, “Omics,” New Technologies, and Tools – Gallatin 12 p.m. Room 10-10:30 a.m. Refreshment Break – Lower Atrium Oral presentation drop-off – Lower Atrium

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4 28th Meeting of the American Society for Rickettsiology

June 11-14, 2016 Big Sky Resort, Big Sky, Montana

Oral presentations will be held in the Gallatin and Jefferson Rooms

Poster presentations will be held in the Madison Room

Funding for this conference was made possible [in part] by R13 AI126727-01 from the National Institute of Allergy and Infectious Diseases. The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the U.S. Department of Health and Human Services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government.

5 Saturday, June 11th

3:00 – 6:45 p.m. Conference Registration & Check-In – Lower Atrium

6:00 – 8:00 p.m. Welcome Reception – Huntley Dining Room Hors d’oeuvres and two drink tickets per guest provided to be used at the full cash bar

7:00 – 8:00 p.m. Invited Historical Lecture – Huntley Dining Room Dr. Jorge Benach – “From Spirochetes to Rickettsia: The Trail-blazing Career of Willy Burgdorfer”

Sunday, June 12th

7:15 – 8:15 a.m. Hot Breakfast Buffet – Huntley Dining Room

7:15-8:15 a.m. Oral presentation check-in and drop-off – Registration Desk, Lower Atrium

8:15 – 9:00 a.m. Howard Ricketts Memorial Invited Lecture – Gallatin Room Dr. Erol Fikrig – “I Don’t Believe in Vaccines”

9:00 – 9:40 a.m. Plenary Presentation- Gallatin Room Dr. Yasuko Rikihisa – “How do Ehrlichia Enter and Thrive in Phagocytes?”

9:40 – 10:00 a.m. Coffee Break – Lower Atrium

Oral presentation drop-off – Registration Table, Lower Atrium Poster Session 1 set-up – Madison Room

6 10:00 a.m. – Noon Concurrent Sessions – Scientific Session 1: Epidemiology, Ecology, and Emerging 2: Coxiella/Bartonella Pathogenesis Diseases Session Chairs: Jim Samuel and Stacey Gilk

Session Chairs: Kevin Macaluso and Andrea Varela-Stokes

Gallatin Room Jefferson Room 10:00 a.m. Abstract #1: Unraveling the Biology of Abstract #8: Searching for In vitro and Emerging Rickettsial Pathogens In vivo Phenotypes for Coxiella burnetii Kevin R. Macaluso* Pathogenesis James E. Samuel*, Erin van Schaik, Elizabeth Di Russo Case, Sara Talmage, Mary Weber

10:25 a.m. Abstract #2: Dynamics of Amblyomma Abstract #9: Cholesterol and Loss of the maculatum-associated Rickettsiae at Intracellular Niche Individual Tick and Population Levels Stacey Gilk* Andrea S. Varela-Stokes*

10:45 a.m. Abstract #3: Rediscovery of a Pathogenic Abstract #10: Subversion of Macrophage Spotted Fever Group Rickettsia Species Autophagy and Inflammatory Pathways by Associated with the Tick Dermacentor Coxiella burnetii parumapertus in the Western U.S. Caylin G. Winchell*, Joseph G. Graham, Christopher Paddock*, Michelle Allerdice, Richard C. Kurten, Daniel E. Voth Sandor Karpathy, Michael Levin, Alyssa Snellgrove, Jana Ritter, Travis Smith, Tom Becker, Jerome Goddard

11:00 a.m. Abstract #4: Scrub Typhus in Africa Abstract #11: Developmental Alice N. Maina*, Ju Jiang, Katherine C. Transcriptome of Coxiella burnetii Reveals Horton, Jacqueline W. Thiga, Jeremiah a Role for Cell Wall Modification in Laktabai, Thomas Holland, Ammar A. Formation of the Small Cell Variant Ahmed, John Waitumbi, Guillermo Kelsi M. Sandoz*, David L. Popham, Paul A. Pimentel, Wendy P. O’Meara, Allen L. Beare, Daniel E. Sturdevant, Vinod Nair, Richards Robert A. Heinzen

11:15 a.m. Abstract #5: Global Presence of Rickettsia Abstract #12: Striae in Human Skin felis and Rickettsia felis-like Organisms Associated with Bartonella henselae Allen L. Richards*, Alice N. Maina, Heidi K. Infection St. John, Ju Jiang, Christina M. Farris Marna Ericson*, Azar Maluki, Rosalie Greenberg, Erin Dodd, John Williams

11:30 a.m. Abstract #6: Novel Anaplasma Species in Abstract #13: Horizontally Acquired tRNA the Environmentally Threatened Florida and Metabolic Genes Fortify Coxiella Gopher Tortoise burnetii’s Physiology Francy L. Crosby*, Liliet Peltierra, Amy L. Rahul Raghavan*, Abraham S. Moses, Jess A. Weeden, James F.X. Wellehan, Mary B. Millar, Matteo Bonazzi, Paul A. Beare Brown, Anna M. Lundgren, Anthony F. Barbet

7 11:45 a.m. Abstract #7: Rickettsial Infections in Native Abstract #14 The Pangenome and Variome Rodents and Insectivores in Northwestern of Coxiella burnetii Georgia Dimitrios Frangoulidis*, Mathias C. Walter Audrey E. Osterbind*, Bryan N. Ayres, Jamie L. Perniciaro, William L. Nicholson

Noon – 1:20 p.m. Buffet Luncheon – Huntley Dining Room

1:30 – 2:45 p.m. Poster Session 1 – Madison Room Refreshments and Cash Bar Provided

2:30 – 3:00 p.m. Oral presentation drop-off – Registration Desk, Lower Atrium

Madison Room Abstract #15: Serology Study of Q-fever in Cattle and Small Ruminants in Georgia E. Mamisashvili*, M. Donduashvili, K. Goginashvili, T. Tigilauri, L. Gelashvili, O. Parkadze, L. Avaliani, L. Ninidze, I. Menteshashvili, M.J. Stuckey, B.B. Chomel Abstract #16: Complete Tick Mitochondrial Genomes from the Ion Torrent PGM Gregory A. Dasch*, Zachary C. Holmes, T. Brian Shirey, Jasmine R. Hensley, Maria L. Zambrano

Abstract #17: First Report of Theileria cervi Infection in Roe Deer (Capreolus capreolus) in the Republic of Korea Kyoung-Seong Choi*, Yu-Jung Han, Joon-Seok Chae, Do-Hyeon Yu, Jinho Park, Bae-Keun Park, Hyeong- Chul Kim

Abstract #18: Evaluation of Gulf Coast Ticks (Acari: Ixodidae) for Ehrlichia and Anaplasma Species Michelle Allerdice*, Joy Hecht, Sandor Karpathy, Christopher Paddock

Abstract #19: Knowledge of Rickettsiosis among First Contact Physicians from a Rural Community in Mexico César Lugo-Caballero*, Karla R. -Rosado, Irving Dzul-Tut,Angel Balam-May, Juan José Arias-León Jorge Zavala-Castro

Abstract #20: Clinical Case: Fatal Rickettsiosis in Southern Mexico César Lugo-Caballero*, Karla R. Dzul-Rosado, Georgina Rodríguez-Moreno, Karina López-Avila, Raúl Tello- Martín, Jorge Zavala-Castro

Abstract #21: Production and Evaluation of Candidates for the Development of DNA Vaccines against R. rickettsii Karla R. Dzul-Rosado*, Cesar Lugo-Caballero, Javier Balam-Romero, Jorge Zavala-Castro Abstract #22: An Ecological Analysis of the Bacterial Microbiome of Dermacentor andersoni in Populations with Differing Vector Competence Cory Gall*, Glen Scoles, Krisztian Magori, Kelly Brayton Abstract #23: Phylogeography of Orientia tsutsugamushi Amy Fleshman*, Crystal M. Hepp, Kristin E. Mullins, Jason Sahl, Daniel Paris, Sabine Dittrich, Paul Newton, Allen L. Richards, Talima R. Pearson

8 Abstract #24: Detection of Spotted Fever Group Rickettsiae in Ticks from the Republic of Korea and the Development of Quantitative Real-time Polymerase Chain Reaction Assays for Rickettsia monacensis and Rickettsia sp. LON-13 Ju Jiang*, Jonathan Erber, Allison Dauner, Theron Gilliland Jr, Shuenn-Jue Wu, John Lee, Heung-Chul Kim, Sung-Tae Chong, Won-Jong Jang, Terry Klein, Allen Richards

Abstract #25: Proposed Study on the Prevalence of Coxiellosis among Livestock in Western Azerbaijan M.G. Abdullayev* Abstract #26: Outbreak Investigation of Scrub Typhus in Thailand: 15 Year Experience Wuttikon Rodkvamtook*, Darunee Utennam, Narupon Kuttasingkee, Yutthapong Sudsawat, Chusak Molito, Allen L. Richards, Wei-Mei Ching, Chien-Chung Chao, Piyada Linsuwanon, Jariyanart Gaywee

Abstract #27: EcxR is a Global Gene Regular in Ehrlichia chaffeensis Laxmi U. M. Jakkula*, Huitao Liu, Ying Wang, Roman Ganta Abstract #28: Regulation of Pulmonary Long Non-coding RNAs in a Murine Model of Rocky Mountain Spotted Fever Imran H. Chowdhury*, Abha Sahni, Hema P. Narra, Kamil Khanipov, Yuriy Fofanov, Sanjeev K. Sahni Abstract #29: Neither too Much nor Much Less: Balancing Act of a Promising Drug Target in Rickettsia conorii Jignesh Patel*, Hema Narra, Krishna Mohan, Aishwarya Sahni, Amber Singh, Casey Schroeder, Abha Sahni, Sanjeev Sahni Abstract #30: Biomarkers for Amblyomma maculatum-Tissues and Associated Rickettsiae John V. Stokes*, Gail M. Moraru, Mariola J. Edelmann, Andrea S. Varela-Stokes

Abstract #31: Gene Expression Profiles of the De Novo Folate Biosynthetic Pathway from a Rickettsia endosymbiont In vivo, In ixodes pacificus, and In vitro, in the Ixodes Scapularis ISE6 Tick Cell Line Kristine Teague*, Maryam Alowaysi, Junyan Chen, Jianmin Zhong Abstract #33: Spotted Fever Group Rickettsia Adaptation to the Mammalian Host Sean P. Riley*, Ludovic Pruneau, Juan J. Martinez

Abstract #34: The Coxiella burnetii Effector Cig57 Hijacks FCHO-mediated Vesicular Trafficking Eleanor A. Latomanski*, Patrice Newton, Hayley J. Newton

Abstract #35: Analysis of the Caenorhabditis elegans Innate Immune Response to Coxiella burnetii James M. Battisti*, Lance Watson, Myo Naung, Adam Drobish, Ekaterina Voronina, Michael F. Minnick Abstract #36: Identifying Novel Host Cell Target Proteins of Bartonella Fic Domains Vaughan Trounson*, Joanna Mackichan Abstract #37: Bacteria Transmitted by Ixodes scapularis Ticks Elicit a Novel Immune Deficiency Circuit Dana K. Shaw*, Erin E. McClure, Lindsey J. Brown, Lei Liu, Kathleen DePonte, Alexis A. Smith, Utpal Pal, Erol Fikrig, Joao H.F. Pedra Abstract #38: Experimental Evaluation of Peromyscus ieucopus as a Reservoir Rost of the Ehrlichia muris-like Agent Geoffrey E. Lynn*, Jonathan D. Oliver, Ulrike G. Munderloh

Abstract #39: Tracking Anaplasma Phagocytophilum Infection in Larval Ixodes scapularis Jonathan Oliver*, Geoffrey Lynn, Nicole Burkhardt, Curtis Nelson, Timothy Kurtti, Ulrike Munderloh

9 Abstract #40: Initial Local Inflammatory Response to Ehrlichia muris-like Agent (EMLA) Transmitted by Ticks Tais B. Saito*, Claire Smalley, David H. Walker Abstract #41: Ehrlichia chaffeensis TRP120 Targets Host Proteins that Differentially Influence Intracellular Survival Tian Luo*, Paige S. Dunphy, J. W. McBride Abstract #42: Pathogen Induced Host Cell ER Stress Benefits the Intracellular Bacterium, Orientia tsutsugamushi Kyle G. Rodino*, Lauren VieBrock, Jason A. Carlyon Abstract #43: Identification of Rickettsia rickettsii Type IV Secretion System Effectors Maria Galletti*, L.M. Gaziola, P. Alexandrino, A. Fujita, I. Sauter, M.J.C. Veliz, D. Voth, S. Daffre, M.B. Labruna, A.C. Fogaça Abstract #44: Effect of Rickettsia felis Strain Variation on Infection, Transmission, and Fitness in the Cat Flea, Ctenocephalides felis Sean P. Healy*, Lisa D. Brown, Melana H. Hagstrom, Lane D. Foil, Kevin R. Macaluso

Abstract #45: Immune Responses to Tick Cell-derived Versus Endothelial Culture-derived Ehrlichia muris-like Agent (EMLA) in Mice Patricia A. Crocquet-Valdes*, Tais B. Saito, David H. Walker Abstract #46: Human Monoclonal Antibodies against Tandem Repeat and Outer Membrane Proteins Inhibit Ehrlichia chaffeensis Growth Intracellularly Thangam Sudha Velayutham*, Xiaofeng Zhang, Gopal Sapparapu, Gary Winslow, Nahed Ismail, James E. Crowe, David H. Walker, Jere W. McBride Abstract #47: The Effect of Intranasal Infection with “Less Virulent” Spotted Fever Group Rickettsiae on Subsequent Rickettsia rickettsii Pathogenesis in Guinea Pigs Edward Shaw*, Jeff Gruntmier, Susan Little

Abstract #48: Immunopathological Study of Scrub Typhus in Rhesus Macaques Following Intradermal Inoculation of Orientia tsutsugamushi Karp and Gilliam Strains Piyanate Sunyakumthorn*, Tippawan Anantatat, Rawiwan Im-erbsin, Manutsanun Sumonwiriya, Kesara Chumpolkulwong, Sirima Wongwairot, Ajchara Vongsawan, Susanna J. Dunachie, Matthew D. Wegner, Christine A. Ege, Allen L. Richards, Nicholas P. J. Day, Daniel H. Paris

Abstract #49: Q Fever Seroprevalence in Farmers, Wildlife Personnel and Ruminants in Thailand 2012 Kelly A. Fitzpatrick*, Pawinee Doung-ngern, Pattarin Opaschaitat, DechaPangjai, Pawin Padungtod, Gilbert J. Kersh Abstract #50: Optimization of ACCM for Axenic Growth of Multiple Genotypes of Coxiella burnetii Rachael Priestley*, Gilbert Kersh

Abstract #51: Characterization of RpoS in Biphasic Development and Host Cell Colonization of Coxiella burnetii Derek E. Moormeier*, Diane C. Cockrell, Paul A. Beare, Kelsi M. Sandoz, Robert A. Heinzen Abstract #52: Characterization of PKC Signaling during Coxiella burnetii Infection C. Joel Funk*, Daniel E. Voth Abstract #53: Human Factor H-binding Proteins of Bartonella bacilliformis and Potential Role in Serum Resistance Michael Minnick*, Benjamin Mason, Richard Marconi, Linda Hicks

10 Abstract #54: Robust Growth of Avirulent Phase II Coxiella burnetii in Bone Marrow-derived Murine Macrophages Diane C. Cockrell*, Jeffrey G. Shannon, Shelly J. Robertson, Heather E. Miller, Lara Myers, Carrie M. Long, Robert A. Heinzen Abstract #55: Anaplasma Phagocytophilum AmpB is Exposed on the Cytosolic Face of the Pathogen- Occupied Vacuole and Co-opts Host Cell SUMOylation Aminat T. Oki*, Bernice Huang, Andrea R. Beyer, Levi J. May, Jason A. Carlyon Abstract #56: Flotillins Associated with Cholesterol are Required and Recruited for Intracellular Proliferation of Anaplasma phagocytophilum Yasuko Rikihisa*, Qingming Xiong

Abstract #57: Ehrlichia chaffeensis Nucleomodulin TRP32 is Regulated by Diverse Ubiquitin Modifications during Infection Tierra Farris*, Bing Zhu, Jere W. McBride Abstract #58: The Tick Protein Sialostatin L2 Binds to Annexin A2 and Inhibits NLRC4-Mediated Inflammasome Activation Xiaowei Wang*, Dana K. Shaw, Joao H.F. Pedra

Abstract #59: “MicroRNA Signature” of Human Microvascular Endothelium Infected In vitro with Rickettsia rickettsii Abha Sahni*, Hema P. Narra, Jignesh Patel, Sanjeev K. Sahni

Abstract #60: Mechanisms of Non-canonical Inflammasome Activation by Rickettsia Claire Smalley*, R. Fang, J. Bechelli, D.H. Walker Abstract #61: The Rickettsia conorii Adr2 Promotes Resistance to Complement-mediated Killing Daniel A. Garza*, Sean P. Riley, Juan J. Martinez

Abstract #62: Involvement of Pore Formation and Osmotic Lysis in the Rapid Killing of Gamma Interferon-Pretreated, C166 Endothelial Cells by Rickettsia prowazekii Jenifer Turco* Abstract #63: Autophagy Promotes Rickettsia australis Replication in Primary Murine Macrophages Jeremy Bechelli*, Claire Smalley, Xuemei Zhao, Seungmin Hwang, David H. Walker, Rong Fang

Abstract #64: Post-translational Modification of the Immunodominant Autotransporter, OmpA in Rickettsia rickettsii Nicholas F. Noriea*, Tina R. Clark, Ted Hackstadt

Abstract #65: Characteristics of Unique Rickettsia rickettsii Iowa Clones Tina R. Clark*, Ted Hackstadt, Nick Noriea

11 3:00 – 5:00 p.m. Scientific Session 3: Host-Pathogen Interactions l Session Chairs: Jere McBride and Kyle Rodino

Gallatin Room 3:00 p.m. Abstract #66: Ehrlichia chaffeensis Effector Driven Reprogramming of the Macrophage Jere W. McBride*

3:25 p.m. Abstract #67: Rickettsia parkeri Harnesses Intercellular Tension Forces to Promote Cell-to- cell Spread Matthew D. Welch*, Rebecca L. Lamason, Effie Bastounis, Natasha M. Kafai, Ricardo Serrano, Juan C. del Álamo, Julie A. Theriot

3:45 p.m. Abstract #68: Anaplasma phagocytophilum Parasitizes Host Acid Sphingomyelinase for Completion of its Infection Cycle Chelsea L. Cockburn*, Jason A. Carlyon 4:00 p.m. Abstract #69: Metabolic and Biosynthetic Activity of Ehrlichia chaffeensis in a Host Cell- free Medium Roman R. Ganta*, Vijay K. Eedunuri, Chuanmin Cheng, Anders Omsland, Dan Boyle

4:15 p.m. Abstract #70: E. chaffeensis Induced Wnt-mTOR Signaling Regulates Transcription Factor TFEB to Inhibit Autophagy Taslima T. Lina*, Jere W. McBride, Tian Luo

4:30 p.m. Abstract #71: Targeted Mutagenesis in Ehrlichia chaffeensis by Homologous Recombination Ying Wang*, Chuanmin Cheng, Roman R. Ganta 4:45 p.m. Abstract #72: Monovalent Cation/Sodium: Proton Antiporter Proteins of Ehrlichia chaffeensis Lanjing Wei*, Huitao Liu, Roman R. Ganta

5:15 – 6:15 p.m. ASR Business Meeting and Election of New Officers – Jefferson Room

7:00 – 9:00 p.m. Gala Dinner-Lone Peak Pavillion A full cash bar will be available where guest may use any remaining drink tickets Guest dinner tickets may be purchased at the registration table in the Lower Atrium until 6:30 p.m.

12 Monday, June 13th

7:00 – 8:00 a.m. Hot Breakfast Buffet – Huntley Dining Room

Oral presentation drop-off – Registration Table, Lower Atrium Poster Session 2 set-up – Madison Room

8:00 – 10:00 a.m. Concurrent Sessions – Scientific Sessions

4: Vector Biology & Vector-Pathogen 5: Infection, Diagnosis, and Treatment Interactions Session Chairs: Daniel Paris and Cecilia Kato Session Chairs: Michael Levin and Girish Neelakanta

Gallatin Room Jefferson Room 8:00 a.m. Abstract #73: Infection-Derived Lipids Elicit Abstract #80: The Burden of Tropical a Novel Immune Deficiency Circuitry in Rickettsioses – the Asian Perspective Arthropods Daniel Paris* Joao Pedra*

8:25 a.m. Abstract #74: One of the Good Catch! Abstract #81: Vascular Permeability with Fishing Cector to Study Tick-pathogen Rickettsial Infections is Abrogated by Interactions Membrane-Active Chelators Girish Neelakanta* J. Stephen Dumler*, Valeria Pappas-Brown, Emily G. Clemens, Dennis J. Grab 8:45 a.m. Abstract #75: Bartonella quintana Utilizes a Abstract #82: A Candidate Vaccine Induces Protein Partner Switch Mechanism to Cellular and Humoral Immunity and Survive Stressful Arthropod Protects from Tick-transmitted Ehrlichia Conditions chaffeensis Infection in a Canine Host Jane E. Koehler*, Henriette Macmillan, Sally Jodi L. McGill*, Arathy D.S. Nair, Chuanmin Lyons‐Abbott, David M. Dranow, Gina M. Cheng, Rachel A. Rusk, Deborah Jaworski, Borgo, James W. Fairman, Stephanie J. Roman R. Ganta Huezo, Donald D. Lorimer, Bart L. Staker, Robin Stacy, Stephanie Abromaitis, Thomas E. Edwards, Peter J. Myler

9:00 a.m. Abstract #76: De novo Folate Biosynthesis Abstract #83: An Intradermal Inoculation of Rickettsia Endosymbiont Ixodes Mouse Model for Immunological pacificus, a New Rickettsia Species Isolated Investigations of Acute Scrub Typhus and from Ixodes pacificus Persistent Infection Jianmin Zhong*, Maryam Alowaysi, Jimmy Lynn Soong*, Nicole L. Mendell, Juan P. Bodnar, Kristine Teague, Junyan Chen Olano, Dedeke Rockx-Brouwer, Guang Xu, Yenny Goez-Rivillas, Claire Drom, Thomas R. Shelite, Gustavo Valbuena, David H. Walker, Donald H. Bouyer 13 9:15 a.m. Abstract #77: Molecular Mechanism of Wolbachia Induced Cytoplasmic Incompatibility John F. Beckmann*, Judith A. Ronau, Mark Hochstrasser

9:30 a.m. Abstract #78: Effects of R. Amblyommii Abstract #85: Prospective Assessment of Infection on the Vector Competence of the Etiology of Illness after a Tick Bite in Amblyomma Americanum Ticks for R. Northeastern China rickettsii Na Jia*, Xue-Bing Ni, Jia-Fu Jiang, Yuan-Chun Michael L. Levin*, Alyssa Snellgrove Zheng, Qiu-Bo Huo, Bao-Gui Jiang, Rui-Ruo Jiang, Lan-Ma, Wu-Chun Cao

9:45 a.m. Abstract #79: Rickettsia parkeri and Abstract #86: Diagnostic Assessment of “Candidatus Rickettsia Andeanae” Tropism Testing Blood Verses Serum from Acute in Tissues of Experimentally Infected RMSF Patients Amblyomma maculatum (Gulf Coast Tick) Cecilia Kato*, Gerardo Alvarez, Ida Chung, Jung Keun Lee*, Gail M. Moraru, Amanda B. Christopher Paddock, Enrique Bolado Harper, John V. Stokes, Haley Parker, Jacob Hughes, Andrea S. Varela-Stokes

10:00 a.m. – Noon Poster Session 2 – Madison Room Refreshments Provided

2:30 – 3:00 p.m. Oral presentation drop-off – Registration Desk, Lower Atrium

Madison Room Abstract #87: Theileria buffeli Infections in Grazing Cattle Located in Mountainous Areas of the Republic of Korea Kyoung-Seong Choi*, Joon-Seok Chae, Do-Hyeon Yu, Jinho Park, Bae-Keun Park, Hyeong-Chul Kim

Abstract #88: Small Mammals as Hosts of Ehrlichia muris eauclairensis and Other Rickettsial Pathogens in Eau Claire, Wisconsin Bryan N. Ayres*, Audrey E. Osterbind, Jamie L. Perniciaro, Joy A. Hecht, William L. Nicholson Abstract #89: Serological Survey of Canine Tick-borne Infections Using Species-specific Serological Markers Jiayou Lui*, M. E. McCown, C. Fiorello, D. Scorpio, M. Filipovic, J. Saucier, B. Thatcher, R. Chandrashekar Abstract #90: Consistencies in Spatio-Temporal Trends, and Climatic and Other Geospatial Risk Factors for Rickettsial Diseases in the Central Midwestern United States Ram K. Raghavan*, Gary A. Anderson, Doug. G. Goodin, Roman R. Ganta

Abstract #91: A Distributional Survey of Ticks (Acari: Ixodida) in Georgia, USA William L. Nicholson* Abstract #92: Spotted Fever Group Rickettsiae in Baltic Region Algimantas Paulauskas*, Jana Radzijevskaja, Asta Aleksandraviciene

Abstract #93: Spotted Fever Group Rickettsia Species Identified among Ticks in Georgia Ekaterine Zhgenti*, Roena Sukhiashvili, Richard J. Obiso, Christina M. Farris, Ju Jiang, Allen L. Richards

14 Abstract #94: Risk Assessment for Vector-borne Diseases at Gateway National Recreation Area, NY: Monitoring Ticks, Preliminary Study, 2014 Ellen Stromdahl*, Garrett Heck, Mary Vince, Waheed Bajwa, Zahir Shah, David Wong Abstract #95: Evaluating an Ecological Niche Model for Rickettsia montanensis Infected Dermacentor variabilis Based on Ticks Collected from Humans Using Environmentally Captured Ticks Heidi K. St. John*, Penny M. Masuoka, Melissa K. Miller, Melissa L. Adams, Johanna G. Flyer-Adams, Ju Jiang, Patrick J. Rozmajzl, Ellen Y. Stromdahl, Allen L. Richards Abstract #96: Survey for Rickettsia Species in Dermacentor variabilis Ticks Collected from National Parks and Monuments across the Eastern United States Sandor E. Karpathy*, Joy A. Hecht, Tammi L. Johnson, Rebecca J. Eisen, Christopher D. Paddock

Abstract #97: Flea-borne Rickettsiae in Southeastern Georgia, USA Marina E. Eremeeva*, Danielle Capps, Amanda Jo Williams-Newkirk, Gregory A. Dasch, Lorenza Beati, Lance Durden Abstract #98: Prevalence of Q Fever and Rickettsia in Ticks in Northeastern Azerbaijan Rita Ismayilova*, Agarahim Mammadsalahov, Rakif Abdullayev, Esmiralda Seyidova, Sabina Ibrahimova, Elvin Naghiyev

Abstract #99: Coxiella burnetii Small RNA 12 (CbsR12) Targets carA Transcripts In vitro Shaun Wachter*, Indu Warrier, Michael F. Minnick

Abstract #100: Alterations in Dermacentor andersoni Transcription in Response to Anaplasma marginale Infection Susan M. Noh*, Forgivemore Magunda, Sebastian Aguilar, Kelly A. Brayton Abstract #101: Characterization of Small, Noncoding RNAs in Rickettsia prowazekii Casey L. C. Schroeder*, Hema P. Narra, Mark Rojas, Abha Sahni, Kamil Khanipov, Jignesh Patel, Yuriy Fofanov, Sanjeev K. Sahni

Abstract #102: Metagenomics of Head Lice Using the Ion Torrent PGM Gregory A. Dasch*, Zachary C. Holmes, T. Brian Shirey, Marina E. Eremeeva, Sarah Zohdy Abstract #103: Decoding the Post-transcriptional Regulatory Networks in Rickettsia conorii Hema P. Narra*, Casey L.C. Schroeder, Abha Sahni, Yuriy Fofanov, Sanjeev K. Sahni

Abstract #104: Cytoskeleton Interactions with the Coxiella burnetii Parasitophorous Vacuole Heather Miller*, Charlie Larson, Robert Heinzen Abstract #105: Examination of Tick Vectorial Capacity Factors during Tick-borne Bacterial Infection Kathryn E. Reif*, Jessica K. Ujzco, Deb C. Alprin, Susan M. Noh

Abstract #106: Nuclear Trafficking of the Anti-apoptotic Coxiella burnetii Effector Protein AnkG Requires Binding to p32 and Importin-α1 Walter Schäfer*, Rita A. Eckart, Benedikt Schmid, Kerstin Hof, Yves Muller, Bushra Amin, Anja Lührmann

Abstract #107: Effects of Novel Bacteriophage on Abalone Exposed to Candidatus Xenohaliotis californiensis – The Causative Agent of Abalone Withering Syndrome James D. Moore*, Ashley Vater, Barbara A. Byrne Abstract #108: Ehrlichia chaffeensis TRP120 Effector Autoubiquitinates and Contains a C-terminal HECT E3 Ligase Domain Bing Zhu*, Tierra R. Farris, Seema Das, Jere W. McBride

15 Abstract #109: Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Directly Regulates Host Gene Expression Tierra Farris*, Bing Zhu, Jere W. McBride Abstract #110: Molecular Detection of Rickettsia Species in Ticks and Fleas in Eastern Missouri and Southern Illinois Catherine Santanello*, Redir Barwari

Abstract #111: Molecular Survey of Arthropod Vectors for Rickettsia in the Eastern Thailand Darunee Utennam*, Kamonwan Siriwatthanakul, Chakrit Hirunpetcharat, Wuttikorn Rodkvamtook, Charnchudhi Chanyasanha, Pimmada Sakpisal, Narupon Kuttasingkee, Yutthapong Sudsawat, Chanakan Suwanbongkot, Jariyanart Gaywee

Abstract #112: Rickettsia cf sp. Infection in Thailand Jariyanart Gaywe*, Wuttikon Rodkvamtook, Walairat Pronwiroon, Darunee Utainnam, Piyanate Salyakumthorn, Pimmada Sakpisal, Narupon Kuttasingkee, Kamonwan Siriwatthanakul, Chanakan Suwanbongkot, Allen L. Richards, Suradet Jaruchinda Abstract #113: Interaction of Rickettsia felis and Wolbachia endosymbionts in Cat Fleas, Ctenocephalides felis Krit Jirakanwisal*, Supanee Hirunkanokpun, Kevin R. Macaluso Abstract #114: Mechanical Infection of Leptotrombidium spp. Mites with Human Pathogenic Strains of Orientia tsustsugamushi Piyada Linsuwanon*, Elizabeth Wanja, Surachai Leepitakrat, Taweesak Monkana, Piyanate Sunyakumthorn, Daniel Paris, Silas Davidson, Opas Thachin, Allen Richards, James Jones

Abstract #115: Investigations into the Dynamics of Coxiella burnetii Infections and Possible Vaccination Options to Control the Disease in Intensively-reared Goats John Stenos*, Michael Muleme, Gemma Vincent, Alexander Cameron, Joanne Devlin, Angus Campbell, Colin Wilks, Stephen Graves, Mark Stevenson, Simon Firestone

Abstract #116: Comparison of Lethal and Nonlethal Mouse Models of Orientia tsutsugamushi Reveals T-cell Population-associated Cytokine Signatures Correlated with Disease or Protection Alison Luce-Fedrow*, Suschsmita Chattopadhyay, John B. Patton, Allen L. Richards Abstract #117: Disease Course and Immune Response of Scrub Typhus in Rhesus Macaques to Increasing Doses by Intradermal Inoculation of Orientia tsutsugamushi Piyanate Sunyakumthorn*, Manutsanun Sumonwiriya, Rawiwan Im-erbsin, Tippawan Anantatat, Kesara Chumpolkulwong, Sirima Wongwairot, Ajchara Vongsawan, Susanna J. Dunachie, Eric D. Lombardini, Robin L. Burke, Allen L. Richards, Nicholas P. J. Day, Daniel H. Paris Abstract #118: DNA Vaccine Candidates Conjugated with T-cell Memory Enhancer to Study its Effectiveness for Long Term Protection Wei-Mei Ching*, Hua-Wei Chen, Zhiwen Zhang, Chye Chan, Chien-Chung Chao

Abstract #119: Antibiotic Efficacy against Coxiella burnetii in Axenic Media Gilbert J. Kersh*, Charles Evavold Abstract #120: Development of Highly Sensitive Quantitative PCR for Setection of Bartonella bacilliformis by Targeting a Multiple-copy DNA Sequence Hua-Wei Chen*, Ai Mochida, Philip Ching, Chien-Chung Chao, Wei-Mei Ching Abstract #121: Molecular Diagnostics of Q-Fever at the Laboratory of the Ministry of Agriculture M. Kokhreidze*, N. Vepkhvadze, M. Donduashvili, E. Mamisashvili, L. Gelashvili, M.J. Stuckey, B.B. Chomel 16 Abstract #122: Development and Clinical Validation of Real-time PCR Assays for the Detection of Ehrlichia Species in Human Specimens C.Y. Kato*, I.H. Chung, A.L. Austin Abstract #123: Rapid Diagnostic Test for Antibody with Korean Isolated Anaplasma Phagocytophilum from Apodemus agrarius Joon-Seok Chae*, Jun-Gu Kang, Sung-Suck Oh, Miyoung Yang, Sungjae Kim, Jeong-Byoung Chae, Yong- Sun Jo, Yun-Kyung Cho, Do-Hyeon Yu, Jeongmi Kim Abstract #124: Molecular and Serological Evidence of Anaplasmosis in South Korea Seon Do Hwang*, Seung Hun Lee, Hae Kyung Lee, Yeong Seon Lee

Abstract #125: Clinical Characteristics of Rickettsiosis in Southern Mexico César Lugo-Caballero*, Karla R. Dzul-Rosado, Adolfo Palma-Chan, Jorge Zavala-Castro Abstract #126: Social Intervention against Tick-borne Diseases in a Rural Community of Mexico Karla R. Dzul-Rosado*, César Lugo-Caballero, Juan Jose Arias-Leon, Gaspar Peniche-Lara, Jorge Zavala- Castro

Abstract #127: Generation of Humanized Chimeric IgG and IgM Antibodies Specifically Recognize the 56 kDa Protein Antigen from Orientia tsutsugamushi Chien-Chung Chao*, Zhiwen Zhang, Tatyana Belinskaya, Wei-Mei Ching Abstract #128: Importance of Geographic Relevance in Determining the Diagnostic Cut-off for Scrub Typhus Serology Jamie L. Perniciaro*, Alfred T. Harding, William L. Nicholson Abstract #129: Enrichment of Rickettsia for Detection and Increased Sensitivity of Diagnostic Samples Cecilia Kato*, Marah Condit

Abstract #130: Experimental Evolution of Coxiella burnetii Using Chemostats James M. Battisti*, Margie Kinnersley, Michael F. Minnick Abstract #131: Anaplasma phagocytophilum 16S rDNA Sequences Present in Domestic Dogs from the Mnisi Community Area, Mpumalanga Province, South Africa Mamohale E. Chaisi, Samantha K. Wills, Agatha O. Kolo*, Kelly A. Brayton, Marinda C. Oosthuizen

Abstract #132: High-content Imaging Reveals Increased Cellular Endocytosis during Coxiella burnetii Parasitophorous Vacuole Maturation Charles L. Larson*, Diane C. Cockrell, Robert A. Heinzen Abstract #133: Ehrlichia chaffeensis Effector TRP47 Binds Host DNA and is SUMOylated. Clayton E. Kibler*, Sarah L. Milligan, Tierra R. Farris, Bing Zhu, Jere W. McBride

Abstract #134: Structure and Distribution of VirB6-4 in A. phagocytophilum Francy L. Crosby*, David R. Allred, Yu-Ping Xiao, Donna S. Williams, Anna M. Lundgren, Anthony Barbet

Abstract #135: Ehrlichia chaffeensis TRP120 Influences Notch Signaling and Cell Cycle by Modulating the SCF Ubiquitin Ligase FBW7 to Promote Infection Jennifer Y. Wang*, Tian Luo, Paige Dunphy, Jere W. McBride Abstract #136: Protein Aggregation in Ehrlichia chaffeensis during Macrophage Infection Dorota Kuczynska-Wisnik, Michal Zolkiewski, Roman R. Ganta* Abstract #137: Ehrlichia chaffeensis Mediated Epigenetic Reprogramming of the Host Facilitates Intracellular Survival Shubhajit Mitra*, Paige S. Dunphy, Jere W. McBride

17 Abstract #138: Brain Endothelial Activation and CXCR3-Related Gene Expression in Murine Models of Scrub Typhus Thomas R. Shelite*, Nicole L. Mendell, Kathryn C. Smith, David H. Walker, Donald H. Bouyer, Lynn Soong Abstract #158: Spotted Fever Group Rickettsiae Hijack Annexin A2-mediated Host Fibrinolytic Machinery for their Adherence to Vascular Endothelial Surfaces Bin Gong*, Donald H. Bouyer*, Tuha Ha, Nicole Mendell, Yuan Qiu, Thomas Shelite, Guang Xu, Qinyu Gong, Thomas Ksiazek, Jia Zhou, Vsevolod Popov

Free Afternoon to Explore!

The Big Sky Resort offers a wide range of activities and adventures both on the property and across the region. Of course, the area’s main attraction is Yellowstone National Park. Dining, shopping, and spa and wellness options can be found on-site at the Big Sky Resort.

Visits to Yellowstone, plus various expeditions, zip-lining and high-ropes courses, skeet shooting and archery, golf, paintball, fishing, rafting, horseback riding, and other activities are available by contacting:

Basecamp Call: (406) 995-5769 Email: [email protected]

18 Tuesday, June 14th

8:00 – 10:00 a.m. Concurrent Sessions – Scientific Session 6: Host-Vector Pathogen Interactions ll 7: Innate and Adaptive Immunity Session Chairs: Juan Martinez, Mary Weber Session Chairs: Joao Pedra and Rong Fang

Gallatin Room Jefferson Room 8:00 a.m. Abstract #139: An Overview of the Abstract #146: Immunity to Infections Molecular Details Governing SFG Rickettsial Caused by Organisms of the Genus Pathogenesis Rickettsia Juan Martinez* David H. Walker*, Rong Fang 8:25 a.m. Abstract #140: Chlamydia trachomatic Abstract #147: Innate Immune Control of inclusion membrane proteins (Incs) are Coxiella burnetii Infection within required for subversion of the host innate Macrophages response and promote inclusion stability Sunny Shin * , William Bradley, Mark Boyer, Mary Weber*, Jennifer Lam, Nicholas Hieu Nguyen, L. Dillon Birdwell, Janet Yu, Noriea, Ted Hackstadt Juliana Ribeiro, Susan Weiss, Dario Zamboni, Craig Roy 8:45 a.m. Abstract #141: Elucidation of the Abstract #148: Investigating the Roles of T Biochemical Interaction between Rickettsia cell-mediated Immunity during Scrub conorii Adr1 and Human Host Vitronectin Typhus Abigail I. Fish*, S.P. Riley, J.J. Martinez Guang Xu*, Thomas R. Shelite, Nicole L. Mendell, Lynn Soong, Donald H. Bouyer, David H. Walker 9:00 a.m. Abstract #142: Orientia tsutsugamushi Abstract #149: Immunoinhibitory receptors effector Ank9 targets host cell secretion PD-1 and LAG-3 contribute to T-cell pathways via Golgi/ER localization and exhaustion during Anaplasma marginale COPB2 interaction infection Andrea R. Beyer*, Kyle G. Rodino, Lauren Wendy Brown*, Tomohiro Okagawa, Satoru VieBrock, Ryan S. Green, Jason A. Carlyon Konnai, James Deringer, Massaro Ueti, Glen Ueti, Glen Scoles, Shiro Murata, Kazuhiko Ohashi 9:15 a.m. Abstract #143: Efficiencies and Abstract #150: Differences in the Optimization of Targeted Insertional Intracellular Fate of Two Spotted Fever Mutagenesis Using a Group II Intron in Group Rickettsia in Macrophage-Like Cells Rickettsia rickettsii Pedro Curto*, Isaura Simões, Sean Riley, Nicholas Noriea*, Tina Clark, Ted Hackstadt Juan J. Martinez 9:30 a.m. Abstract #144: The First Crystal Structure of Abstract #151: Orientia tsutsugamushi the Soluble Domain of RC1339/APRc from Ankyrin Repeat-containing Proteins Ank1 R. conorii Reveals a Striking Conservation and Ank6 Inhibit the Host NF-κB with the Typical Fold of Retropepsins Proinflammatory Pathway Isaura Simões*, Mi Li, Alla Gustchina, Pedro Sean M. Evans*, Jason A. Carlyon Curto, Rui Cruz, Marisa Simões, Carlos Faro, Juan J. Martinez, Alexander Wlodawer

19 9:45 a.m. Abstract #145: Structural Characterization Abstract #152: Rickettsiologist Paul F. of the Anaplasma marginale Antigenically Zdrodovskyi - Larger than Life and not Just Variant Msp2 Protein for his Famous Book Telmo Graça*, Marta G. Silva, Alla S. Marina E. Eremeeva*, Stanislav N. Shpynov, Kostyukova, Guy H. Palmer Irina V. Tarasevich

10:00 – 10:30 a.m. Refreshment Break – Lower Atrium

Oral presentation drop-off – Registration Table, Lower Atrium

10:30 a.m. – 12:00 p.m. Scientific Session 8: Genetics, “Omics”, New Technologies, and Tools – Gallatin Room Session Chairs: Kelly Brayton and Paul Beare

Gallatin Room

10:30 a.m. Abstract #153: In Depth Analysis of Anaplasma marginale: What we Have Learned from Deep Sequencing Kelly Brayton* 10:55 a.m. Abstract #154: Using Genomics and Genetics to Unravel Phase Variation in Coxiella burnetii Paul Beare*, Robert Heinzen

11:15 a.m. Abstract #155: Genomic Characterization of Orientia tsutsugamushi Isolates from Southeast Asia, the Pacific Islands, Australia, Japan, and Korea Kristin E. Mullins*, Amy Fleshman, Crystal M. Hepp, Jason Sahl, Daniel Paris, Sabine Dittrich, Paul Newton, Talima R. Pearson, Allen L. Richards 11:30 a.m. Abstract #156: Biostatistical Prediction of Genes Essential for Growth of Anaplasma phagocytophilum in a Human Promyelocytic Cell Line Using a Random Transposon Mutant Library Kate O’Conor*, Ulrike G. Munderloh 11:45 a.m. Abstract #157: Analysis of Complete Genome Sequence and Major Surface Antigens of Neorickettsia helminthoeca, Causative Agent of Salmon Poisoning Disease Mingqun Lin*, Katherine Bachman, Zhihui Cheng, Sean C. Daugherty, S. Nagaraj, Naomi Sengamalay, S. Ott, A. Godinez, Luke J. Tallon, Lisa Sadzewicz, Sushma Parankush, Claire Fraser, Julie C. Dunning Hotopp, Yasuko Rikihisa

28th Meeting of the American Society for Rickettsiology is Adjourned

20

th 28 Meeting of the

American Society for Rickettsiology

Sunday June 11, 2016

Dr. Howard Taylor Ricketts

Memorial Invited Lecture

Plenary Presentation

Abstracts #1-72

Epidemiology, Ecology, and Emerging

Diseases

Coxiella/Bartonella Pathogenesis

Poster Session 1

Host-Pathogen Interactions l

Funding for this conference was made possible [in part] by R13 AI126727-01 from the National Institute of Allergy and Infectious Diseases. The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the U.S. Department of Health and Human Services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government.

21 Howard Ricketts Memorial Invited Lecture I Don’t Believe in Vaccines Dr. Erol Fikrig von Zedtwitz Professor of Medicine, Yale University Investigator, Howard Hughes Medical Institute

Saliva contains diverse proteins that help an arthropod to engorge on a mammalian host. Some of these molecules may directly, or indirectly, enable pathogen transmission. These interactions are highlighted, using the Lyme disease agent, Borrelia burgdorferi, and Ixodes scapularis ticks, as the paradigm. Work with another tick-transmitted agent, Anaplasma phagocytophilum is also considered, and the paradigm is extended to mosquito-borne infections. Understanding these relationships may lead to new vaccines that are not centered on conventional “pathogen-based” approaches.

Plenary Presentation How Do Ehrlichia Enter and Thrive In Phagocytes? Dr. Yasuko Rikihisa Veterinary Biosciences, Ohio State University

Obligatory intracellular bacteria, such as Ehrlichia chaffeensis, perish unless they can enter eukaryotic cells. Ehrlichia uses the C-terminus of the outer-membrane invasin, Entry-triggering protein of Ehrlichia (EtpE; EtpE-C), which directly binds the mammalian cell-surface glycosylphosphatidyl inositol–anchored protein, DNase X. Our subsequent study showed that EtpE-C binding to DNase X drives Ehrlichia entry by engaging a mammalian cell-surface glycoprotein CD147 and cytoplasmic hnRNP-K and activating N-WASP–dependent actin polymerization. To survive and proliferate in the membrane-bound inclusion, E. chaffeensis depends mostly on host-synthesized nutrients, as it has a limited number of genes for biosynthesis and metabolism. Our study found that ehrlichia replicates in an early amphisome which contains RAB5A/B/C and an autophagy initiation protein, ATG5. Also, ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1, and ATG5. Ehrlichia acquired host cell pre- incorporated amino acids in a PtdIns3K-dependent manner. Moreover, RAB5 siRNA treatment, or overexpression of a RAB5-specific GTPase-activating protein or of dominant-negative RAB5 inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating PtdIns3K complex, VPS34 and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated cellular PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and PtdIns3K, while avoiding autolysosomal killing.

Abstract #1 Unraveling the biology of emerging rickettsial pathogens Kevin R. Macaluso Vector-borne Disease Laboratories, Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University

There has been a recent relative increase in reported rickettsial infections in the United States. Coinciding with this increase has been the discovery of novel associations between vectors and Rickettsia species, newly recognized rickettsial pathogens, and identification of transmission potential of what have been considered to be rickettsial symbionts. Thus, the transmissibility of rickettsiae, vectorial capacity, and the classification of rickettsial pathogens warrant further attention. In addition to the dynamic ecology of tick-borne rickettsial diseases, an emerging flea-borne rickettsiosis, caused by Rickettsia felis, is an example of an understudied pathogen. Originally identified in the United States as a human pathogen in 1991, it is now recognized that human infections caused by R. felis account for cases of fever in sub-Saharan Africa. The cosmopolitan distribution of this pathogen is credited to the equally widespread occurrence of cat fleas (Ctenocephalides felis), the primary vector and reservoir of R. felis. Similar to tick-borne rickettsiae, limited knowledge of 22 basic vector-pathogen interactions continue to hinder research relative to transmission biology, ecology and epidemiology of the diseases.

Abstract #2 Dynamics of Amblyomma maculatum-associated rickettsiae at individual tick and population levels Andrea S. Varela-Stokes Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University

Influence of non-pathogenic rickettsiae on movement, maintenance and transmission of sympatric pathogenic rickettsiae in ticks is not well-understood. Further, how other bacterial genera within the tick microbiome affect transmission of pathogens at individual tick and population levels, is unclear. Our laboratory uses Amblyomma maculatum (Gulf Coast tick) to explore movement of the pathogen, Rickettsia parkeri, and non-pathogen, “Candidatus Rickettsia andeanae,” in the tick and vertebrate host. Using adult ticks infected with R. parkeri, “Ca. R. andeanae”, co-infected, or uninfected, and fed on rabbit hosts, we consistently found “Ca. R. andeanae” by qPCR in salivary glands and midgut from “Ca. R. andeanae” and co-infected groups on Days 0, 6 and 12. Rickettsia parkeri was rarely detected, though levels were highest in salivary glands from the co-infected group at Day 12, when presence is not likely biologically important for horizontal transmission. To confirm rickettsial presence in tick tissues, we used transmission electron microscopy and fluorescence microscopy, with labeled antibodies and riboprobes, on samples with highest rickettsial levels. Blood samples and selected tissues from rabbits were negative by qPCR, though rickettsial exposure was evident based on serological testing, demonstrating rickettsial transmission. In co-feeding studies, using R. parkeri-infected adult A. maculatum and recipient nymphs infected or uninfected with “Ca. R. andeanae” on cattle, transmission of R. parkeri was evident only in uninfected nymphs, suggesting populations of “Ca. R. andeanae”-infected ticks may not easily acquire infections with R. parkeri. To further understand rickettsial dynamics inside the tick, we performed proteomics on midgut and salivary gland tissues, selected proteins expressed at higher levels in each tissue, and generated monoclonal antibodies; a similar approach is being pursued to identify biomarkers for A. maculatum rickettsiae. Future studies will explore the impact of other tick microbes, including species of known or unknown pathogenicity, on pathogen maintenance and transmission.

Abstract #3 Rediscovery of a pathogenic spotted fever group Rickettsia species associated with the tick Dermacentor parumapertus in the western United States Christopher Paddock*1, Michelle Allerdice1, Sandor Karpathy1, Michael Levin1, Alyssa Snellgrove1, Jana Ritter2, Travis Smith3, Tom Becker4, and Jerome Goddard5 1Rickettsial Zoonoses Branch and 2Infectious Diseases Pathology Branch, Centers for Disease Control and Prevention, Atlanta, GA; 3Texas Parks and Wildlife Department, Black Gap Wildlife Management Area, Alpine, TX; 4Utah Division of Wildlife Resources, Springville, UT; 5Mississippi State University, Starkville, MS

In 1953, investigators at the Rocky Mountain Laboratories in Hamilton, MT, described the isolation of a spotted fever group (SFG) Rickettsia species from pooled samples of Dermacentor parumapertus ticks collected from jackrabbits in northern Nevada. When inoculated intraperitoneally into male guinea pigs, strains of this organism (designated as the parumapertus agent) produced a self-limiting and non-fatal illness characterized by 1-2 days of low-grade fever, accompanied by scrotal swelling, similar to manifestations elicited by other SFG Rickettsia species of lower virulence than Rickettsia rickettsii. Additional isolates of the parumapertus agent were subsequently obtained from adult D. parumapertus collected in the Great Salt Lake Desert of western Utah and from northern California. Several decades later, investigators characterized many of these isolates by using mouse serotyping methods and determined that these represented a distinct rickettsial serotype, related most closely to the pathogenic species Rickettsia parkeri and Rickettsia sibirica; nonetheless, this SFG Rickettsia species was not further characterized and has remained unnamed and unstudied for approximately the last 50 years. Unfortunately, no extant isolates of the parumapertus agent exist in any rickettsial culture collection around the world, precluding a contemporary phylogenetic placement of this enigmatic species. Herein, we describe efforts to locate Rickettsia-infected D. parumapertus ticks in nature and subsequent cell culture isolation, molecular characterization, and phylogenetic placement of the parumapertus agent, as well as its pathogenic behavior in

23 guinea pigs. Additionally, we consider the potentialities of past and present influences of this distinct SFG Rickettsia on the ecology and epidemiology of rickettsial diseases in the western United States.

Abstract #4 Scrub Typhus in Africa Alice N. Maina1*, Ju Jiang1, Katherine C. Horton2, Jacqueline W. Thiga3, Jeremiah Laktabai4, Thomas Holland5, Ammar A. Ahmed6, John Waitumbi3, Guillermo Pimentel2, Wendy P. O’Meara4,5, Allen L. Richards1 1Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland, 2Naval Medical, Research Unit No. 3, Cario, Egypt, 3Walter Reed Project, Kenya Medical Research Institute, Kisumu, Kenya, 4Moi, University College of Health Sciences, Eldoret, Kenya, 5Duke Global Health Institute & Duke University School of Medicine, Division of Infectious Diseases, Durham, North Carolina, 6Ministry of Health, Djibouti

Scrub typhus is a vector-borne febrile illness caused by Orientia spp. Apart from a few anecdotal reports of suspected cases of scrub typhus in travelers who visited Cameroon, Democratic Republic of Congo and Burundi, information on scrub typhus is fragmentary in Africa. Three independent studies were conducted in Africa to establish whether scrub typhus contribute to the burden of febrile illnesses. Human sera collected from abattoir workers in Djibouti (NAMRU-3) and fever patients in 8 hospitals in 6 ecoregions of Kenya (USAMRU-K) were assessed for presence of IgG antibodies-specific to Orientia spp. Moi University, Kenya in collaboration with Duke University and NMRC conducted a hospital based study on children aged between 1-12 years for evidence of various pathogens including Orientia spp. Screen positive sera were tested in parallel with their corresponding acute-phase sera by serial dilution, to assess for presence of enhanced sero- reactivity, defined as seroconversion or fourfold rise in antibody titer between the acute and convalescent phase sera. Samples that showed enhanced sero-reactivity for antibodies to Orientia spp. by ELISA, were further confirmed by Western blot assays utilizing the recombinant proteins Kpr56 and Kpr47b. IgG antibodies to Orientia spp. were identified in 5% (67/1401) fever patients in Kenya, in 6% (3/49) abattoir workers in Djibouti and in 5.8% (21 /364) of the children aged 1-12 years in western Kenya. Additionally, enhanced sero-reactivity indicative of acute infections was observed in 3.5% (10/281) of the patients aged 1-12 years in western Kenya, and in 9% (1/11) of the abattoir workers in Djibouti. These results, plus the recent identification of scrub typhus cases in Chile and UAE, add evidence to the expansion of the known endemic area of scrub typhus. This therefore, highlights the urgent need for surveillance studies to define the circulating Orientia genotypes, vectors and their vertebrate hosts in these locations.

Abstract #5 Global Presence of Rickettsia felis and Rickettsia felis-like Organisms Allen L. Richards1, Alice N. Maina1, Heidi K. St. John1, Ju Jiang1, Christina M. Farris1,2. 1 Naval Medical Research Center, Silver Spring, Maryland, USA 2 Naval Medical Research Unit #2, Phnom Penh, Cambodia

Following the identification in cat fleas (Ctenocephalides felis) from United States in 1990, Rickettsia felis has been detected in over 30 countries, and human infections have been reported in 18 countries from all continents except Antarctica. One decade later (2003), the first Rickettsia felis-like organisms (RFLOs), Rickettsia sp. RF2125 and Rickettsia sp. RF31, were reported following their collections from near the Thailand–Myanmar border. Subsequently many RFLOs have been described, including the well-characterized Candidatus Rickettsia asemboensis and Ca. Rickettsia senegalensis. R. felis and the RFLOs are genetically closely related and are members of the same clade within the Transition Group of rickettsiae. These agents are commonly associated with fleas, but can also be found in other invertebrates such as ticks, mites, tsetse flies, and mosquitoes. R. felis is a known human pathogen, whereas the pathogenicity of the RFLOs is unknown. The distribution of these agents is similar to the distribution of other rickettsiae, restricted only by their invertebrate hosts. Thus, many of the fleas commonly inhabited by these agents have worldwide distribution so it would be assumed that the agents also have worldwide distribution. This presentation will provide updated information on the global distribution of and the relationship between R. felis and the RFLOs in locations where they are sympatric.

24 Abstract #6 Novel Anaplasma species in the environmentally threatened Florida gopher tortoise Francy L. Crosby1*, Liliet Peltierra1, Amy L. Weeden2, James F.X. Wellehan1, Mary B. Brown1, Anna M. Lundgren1 and Anthony F. Barbet1 1College of Veterinary Medicine, University of Florida, FL, USA; 2Gribbles Veterinary, Auckland, New Zealand

Members of the family Anaplasmataceae are obligate intracellular bacteria that replicate within membrane bound vacuoles in the cytoplasm of cells in vertebrate and invertebrate hosts. Blood smears from two Florida gopher tortoises (Gopherus polyphemus) presenting with anemia, designated as “Prudence” and “Celes”, had intracytoplasmic inclusions within red blood cells (RBCs). Blood from Prudence was used as inoculum to infect a monolayer of ISE6 tick cells. Diff- Quik slide examination of infected ISE6 cells revealed colonies of bacteria within parasitophorous vacuoles, morphologically similar to A. marginale. Polymerase chain reaction using genomic DNA extracted from infected cultures and primers designed to target nearly the entire 16S rRNA of organisms of the family Anaplasmataceae was performed to determine its taxonomic classification. PCR products were cloned and the 16S rRNA sequences obtained were aligned to 16S rRNA sequences deposited in GenBank from different Anaplasma and Ehrlichia species. A phylogenetic tree was constructed based on the sequence distance method, using the maximum-likelihood algorithm with the Kimura two- parameter model. The newly obtained sequence grouped on a separate branch in the genus Anaplasma. There was 97% 16S nucleotide identity to A. marginale, 96% to A. phagocytophilum, A. ovis and A. platys, 95% to Aegyptianella pullorum and 93% to E. chaffeensis, E. canis, E. ruminantium and E. muris. Gopher tortoise RBC collected from 12 wild populations between 2003 and 2006 in North and Central Florida were available for analysis. DNA extracted from RBC of 40 animals with packed cell volumes (PCV) <20 were screened by PCR for Anaplasma. Interestingly, analysis of 16S rRNA sequences revealed that 14 tortoises were infected with bacteria of the same Anaplasma species isolated from the blood of Prudence. This study reports a novel Anaplasma species in gopher tortoises in Florida. The clinical presentation of significant anemia associated with Anaplasma in an environmentally threatened species could have conservation implications. Its isolation in cell culture will aid further studies to develop diagnostic tests and to investigate potential tick vectors and infectivity for other wildlife and domestic animal species.

Abstract #7 Rickettsial Infections in Native Rodents and Insectivores in Northwestern Georgia Audrey E. Osterbind*, Bryan N. Ayres, Jamie L. Perniciaro, and William L. Nicholson Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, GA

Rocky Mountain spotted fever (RMSF) is reported annually in the southeastern United States. Reports of RMSF in humans are likely due to a spectrum of infections caused by a number of related spotted fever group (SFG) rickettsiae. Recent efforts to find Rickettsia rickettsii in ticks from RMSF endemic areas of the South has not been successful. Thus, there is a critical need for re-assessment of the ecology of these agents in areas of the southeastern United States where RMSF is thought to be endemic to better understand the complex system and how it could contribute to human risk for infection. To this end, we initiated a study in northwestern Georgia to assess small mammal and tick infection with the SFG rickettsiae. Small rodents were trapped daily in Sherman traps for 1-3 day periods at monthly intervals from April- September. Ticks were collected from the animals and from vegetation by flagging. Animals were euthanized, bled, examined for ectoparasites, and necropsied for tissues. Spleens, ears, and flagged ticks were tested by using a real-time PCR assay for SFG rickettsiae. Of the 241 animals collected, species of small mammals included Peromyscus spp., Sigmodon hispidus, Sorex spp., one Glaucomys volans, and one Tamias striatus. PCR testing of spleens was able to detect rickettsiae species in 6/241 mammals, 5 are pending confirmation and one has been confirmed as R. parkeri. Serology screening at a 1/32 dilution identified 90/241 animals as seropositive for SFGR. Additionally, PCR analysis indicated that 18 (13.5%) of the 133 ticks thus far tested were positive for Rickettsia species. Of the positive ticks, 11/18 (61.1%) were infected with R. parkeri. The seven remaining gltA positive ticks were tested by PCR targeting ompA gene of SFG rickettsiae. All seven ticks harbored SFG rickettsiae, and sequence analysis identified 6/7 as “Candidatus Rickettsia andeanae”. The remaining rickettsial species identification is pending.

25 Abstract #8 Searching for in vitro and in vivo phenotypes for Coxiella burnetii pathogenesis James E. Samuel*1, Erin van Schaik1, Elizabeth Di Russo Case1, Sara Talmage1, Mary Weber1,2 1Department of Microbial Pathogenesis and Immunology, Texas A&M HSC, Bryan, TX 77807 2Laboratory of Bacteriology, NIH/NIAID Rocky Mountain Laboratories, Hamilton, MT 59840

Coxiella burnetii is a Gram negative, obligate intracellular pathogen and the etiological agent of acute and chronic Q fever. Comparative animal infection data supports the hypothesis that phylogenetically distinct isolate groups vary in their acute disease potential. Hence, surveys of genetic loci can suggest genes that are essential for pathotype-specific acute disease virulence. The organism utilizes an essential type IVB secretion system (T4SS) to promote intracellular survival, replication and virulence in a mouse model of acute disease. In a large-scale screen of T4BSS substrates, we identified at least 90 ORF that encode potential T4SS effectors. Using transposon mutagenesis, we have determined that many of these T4SS substrates appear to be required for replication in model eukaryotic cells. Additionally, several of these effector mutants replicate with wild-type kinetics in selected cell lines, yet are attenuated in primary bone marrow derived mouse macrophages (BMDM), and/or only are attenuated in vivo in SCID mouse infection. All substrates were evaluated bioinformatically and in several heterologous expression assays to predict function. Specific examples of distinct classes of virulence dependent T4SS substrates for which we are establishing molecular models include: 1) CirA (CBU0041), which is conserved among pathotypes, is essential for intracellular replication in vitro and in vivo, and appears to be essential for CCV development by altering the function of RhoA; 2) CBU1217, which is divergent among pathotypes, is required for replication in BMDM and SCID mouse infection but not Vero cells, and appears to contain an F-box domain that may regulate intracellular ubiquitination; 3) CBU388, which is divergent among pathotypes, required for intracellular replication and SCID mouse infection, traffics through the nucleus and may structurally mimic the nuclear export docking protein, Crm1. Acute pathotypes also encode a functional type IV pilus-like, type II secretion system. Inactivation of essential structural components, pilE or pilD, attenuate replication in BMDM and in SCID mice, but not in J774 mouse macrophages. Expanding the knowledge of secreted effectors enables an increasingly refined understanding of the molecular pathogenesis of acute disease caused by C. burnetii and potentially defines therapeutic and vaccine targets for translational approaches to mitigate acute and chronic disease.

Abstract #9 Cholesterol and loss of the intracellular niche Stacey Gilk Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN

Cholesterol, an essential lipid regulating membrane dynamics, trafficking, and signaling, is emerging as an integral player in host-pathogen interactions. Numerous intracellular bacterial pathogens target mammalian cholesterol as a mechanism to gain entry into host cells, as well as a lipid and nutrient source. Notably, cholesterol is implicated in the pathogenesis of Coxiella burnetii, a highly infectious obligate intracellular bacterium and major cause of culture-negative endocarditis. C. burnetii occupies a unique niche inside the host cell, replicating in a modified acidic phagolysosome, or parasitophorous vacuole (PV). The C. burnetii PV membrane is sterol-rich, and both exogenous and endogenous sources of cholesterol traffic to the PV membrane. However, using our cholesterol-free cell culture model system, we discovered that C. burnetii grows optimally in the absence of host cell cholesterol, with a dose-dependent sensitivity to increasing cholesterol levels. Compared to cholesterol-free cells, PVs in cells with cholesterol were smaller and contained lysed bacteria, which correlated with fewer recoverable viable bacteria. Furthermore, trapping cholesterol in the PV membrane with the lysosomal cholesterol egress inhibitor U18666A also resulted in C. burnetii lysis. C. burnetii’s sensitivity to cholesterol is specific to the intracellular environment, as neither cholesterol or U18666A affected axenic bacterial cultures. To further characterize cholesterol-dependent changes in the PV membrane, we utilized a fluorescent dextran trafficking assay to measure fusion between the PV and host endosomes. We found that PVs with lysed bacteria did not accumulate dextran, suggesting cholesterol alters PV fusogenicity. Current experiments are determining cholesterol-mediated changes in the PV environment that could lead to C. burnetii death.

26 Abstract #10 Subversion of macrophage autophagy and inflammatory pathways by Coxiella burnetii Caylin G. Winchell1*, Joseph G. Graham1, Richard C. Kurten2, and Daniel E. Voth1 1Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR; 2Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR

Coxiella burnetii is the causative agent of human Q fever, a debilitating flu like illness that can progress to chronic disease presenting as endocarditis. After inhalation, C. burnetii is phagocytosed by alveolar macrophages and generates a lysosome-like replication niche known as the parasitophorous vacuole (PV). A type IV secretion system (T4SS) is required for generation of the PV and is one of the pathogen’s few known virulence factors. We previously showed that C. burnetii actively recruits autophagosomes to the PV using the T4SS, but does not alter macroautophagy. Interestingly, the cargo receptor p62 localizes to the PV in a T4SS-dependent manner and co-localizes with LC3. p62 and LC3 interact to select cargo for autophagy-mediated degradation, resulting in p62 degradation and LC3 recycling. In C. burnetii-infected macrophages, p62 is not degraded when cells are starved. We hypothesized that stabilization of p62 was due to activation of the NRF2-KEAP1 pathway, a cytoprotective response to oxidative stress and xenobiotics. Indeed, the NRF2-KEAP1 pathway was activated during infection as evidenced by translocation of NRF2 to the nucleus. A downstream target of the NRF2-KEAP1 pathway is NF- g cascade in C. burnetii pathogenesis is unclear. However, previous studies showed differences in inflammasome activation when macrophages were infected with avirulent or virulent C. burnetii isolates. Therefore, we assessed NF- macrophages infected by avirulent or virulent C. burnetii. Collectively, our studies highlight important host cell signaling pathways exploited by C. burnetii and differences in the innate immune response to differing pathotypes.

Abstract #11 Developmental transcriptome of Coxiella burnetii reveals a role for cell wall modification in formation of the small cell variant Kelsi M. Sandoz1*, David L. Popham2, Paul A. Beare1, Daniel E. Sturdevant3, Vinod Nair4, and Robert A. Heinzen1 1Laboratory of Bacteriology, Rocky Mountain Laboratories, Hamilton, MT; 2Department of Biological Sciences, Virginia Tech, Blacksburg, VA; 3Genomics Unit, Research Technologies Branch, Rocky Mountain Laboratories, Hamilton, MT; 4Electron Microscopy Unit, Research Technologies Branch, Rocky Mountain Laboratories, Hamilton, MT

The bacterial cause of human Q fever, Coxiella burnetii, undergoes is a biphasic developmental cycle that generates large cell variant (LCV) and small cell variant (SCV) forms that are biologically, ultrastructurally, and compositionally distinct. LCVs replicate during the exponential phase of growth while SCVs are stationary phase forms that do not replicate. Based on resistance to physical disruption, SCVs are thought to be responsible for C. burnetii’s noted environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. The most striking transcriptional signature of the SCV was upregulation (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3-3 peptide cross-links in peptidoglycan (PG). 3-3 cross-links result in a shorter average distance between the glycan strands of PG, which is hypothesized to generate an overall stiffer PG structure. Among the cross-links identified by mass spectrometry, L,D transpeptidase upregulation correlated with PG of SCV having 46% 3-3 cross-links as opposed to 16% 3-3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. To gain insight into genetic mechanisms responsible for SCV cell wall cross-linking, we generated mutant strains with deletions in the induced L,D transpeptidases genes cbu0053, cbu0318, or cbu0957. We are currently evaluating these strains for altered growth, ultrastructure, and PG cross-linking patterns with the primary aim of identifying the gene responsible for the 3-3 cross-linking upon transition to SCV. Characterization of the gene(s) responsible for SCV cell wall remodeling will improve our understanding of the C. burnetii developmental cycle and its role in generating resistant SCV.

27 Abstract #12 Striae in human skin associated with Bartonella henselae infection Azar Maluki1, Rosalie Greenberg2, Erin Dodd1, John Williams3, Marna Ericson1* 1Department of Dermatology, University of Minnesota 2Medical Arts Psychotherapy Associates, Summit, New Jersey 3The Skin Care & Surgery Center, Summit, New Jersey

Bartonella spp. can cause prolonged infection in humans and a diverse spectrum of visceral and cutaneous manifestations. We hypothesize that a subgroup of striae, linear streaks in the skin, are the result of interactions of the pathogen Bartonella henselae with fibrillar collagen and/or elastin fibers, in the dermis of the skin. These interactions could disrupt normal deposition and organization of the collagen macrofibrils; resulting in linear dermal scars. Previously we reported clusters of immuno-reactive B. henselae associated with fibrillar dermal collagen in a patient with long-standing Bartonellosis. We report on two patients, P1 and P2, who presented with symptoms of Bartonellosis and non-traditional striae. Typically, stretch marks or striae distensae can form when the body produces excess cortisone, during pregnancy, with fast weight gain, overuse of topical corticosteroids or extreme weight-lifting. In patients with Bartonellosis, the striae are found on body sites that do not typically harbor striae distensae; such as the upper back, forearms and popliteal fossa (area behind the knee) with haphazard directionality of the striae. The Bartonella spp. streaks are likely not due to underlying connective tissue disorders such as Ehler’s-Danlos syndrome. Multi-platform blood sample analysis (Galaxy Diagnostics, Raleigh, NC) revealed B. henselae IFA was 1:32 for P1 and for P2 the IFA was 1:256 for both B. henselae and B. quintana. Eighty to one hundred micron thick sections of skin biopsies from lesional and non-lesional skin were multi- stained with antibodies to 1) B. henselae, 2) lymphatics, 3) blood vessels and a 4) nuclear dye and then imaged with single- and multi-photon laser scanning microscopy (including second harmonic generation imaging (SHG) of fibrillar collagen). Striae sections were strongly immunoreactive for B. henselae and the immunoreactive bacteria were associated with lymphatics and blood vessels. Dermal vasculature was highly disrupted. Surprisingly, immunoreactive B. henselae was detected in non-striae sections, but to a much lesser degree. SHG revealed scar-like collagen structure in striae skin only. It is likely the dermis can serve as a niche for persistent Bartonella spp.. Characterization of non-traditional striae in patients with Bartonellosis using advanced imaging techniques presents a unique opportunity to further elucidate the mechanism of Bartonella spp. infection.

Abstract #13 Horizontally acquired tRNA and metabolic genes fortify Coxiella burnetii’s physiology Abraham S. Moses1, Jess A. Millar1, Matteo Bonazzi2, Paul A. Beare3, Rahul Raghavan1* 1Department of Biology and Center for Life in Extreme Environments, Portland State University, Portland, OR 2CNRS, FRE3689, CPBS, Montpellier, France 3Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institutes of Health

Despite decades of research, the basic biology of Coxiella burnetii, the etiologic agent of acute Q fever and chronic endocarditis, is not fully understood. The pathogen has a unique biphasic life cycle, which includes a metabolically active intracellular form that occupies a large lysosome-derived, acidic vacuole. Coxiella is the only bacterium known to thrive in such a compartment, and this ability is fundamental to its pathogenicity; however, very little is known about genes that are vital to Coxiella’s intracellular growth. This lack of knowledge has hindered both the understanding of its intracellular physiology and the development of better therapies. In order to identify metabolic processes that contribute to Coxiella’s intracellular growth and virulence, we interrogated its genome using a novel evolutionary genomics approach. Our analysis identified numerous metabolic genes that were acquired by Coxiella from the environment, including a tRNAGlu, which is a substrate for heme biosynthesis, a fatty acid biosynthesis operon, a biotin biosynthesis gene, and a tryptophan biosynthesis operon. We generated Coxiella strains in which several of these genes were either deleted or contained transposon insertions. In comparison to wild-type C. burnetii NMII strain, the mutant strains exhibited reduced growth in cell-free medium and/or within eukaryotic cells, confirming their importance to Coxiella’s physiology. We also show that small molecules that block heme and biotin biosynthesis significantly inhibit Coxiella’s growth. Collectively, our study demonstrates that foreign-origin genes boost Coxiella’s physiology, and because several of these genes are important to Coxiella but are not present in humans, they are ideal candidates for the development of new anti-Coxiella agents with

28 low side effects. Furthermore, the use of an alternate tRNA to promote infection represents a novel pathogenic mechanism, and because several other pathogenic bacteria encode multiple tRNAGlu isoacceptors, this strategy could be widespread among human pathogens.

Abstract #14 The Pangenome and Variome of Coxiella burnetii Mathias C. Walter and Dimitrios Frangoulidis* Bundeswehr Institute of Microbiology, Munich, Germany

Analysis of genetic differences (gene presence/absence, nucleotide polymorphisms, insertions/deletions and structural variations) among strains of a bacterial species is crucial to understand molecular mechanisms of pathogenesis and also important for the selection of novel targets for anti-bacterial therapy. Up to date, no such comprehensive comparative genome analysis was done for Coxiella burnetii, even though, there are more than twenty whole genome assemblies and ten raw sequence datasets of different strains available. To address this issue, we constructed the pangenome and variome of 35 strains. We expanded the repertoire of only five complete genomes by one finished reference genome and two newly sequenced genomes. We further constructed assemblies from the raw sequence datasets to rise the number of available near-complete genomes to 35. From these genomes we constructed the core- and accessory genome at the nucleotide and gene/protein level by whole genome alignment methods and positional orthology. Then we performed a whole genome variation analysis and determined unique genomic regions, strain-specific genes, SNPs and InDels, insertion elements and conserved structural elements and summarized it as the variome. The pangenome (as a matrix of the core and accessory gene pool) and the variome data (multiple sequence alignments, SNP and InDel annotation, etc.) is stored in a database and available for download. This resource allowed us to identify factors influencing the different growth behaviour of several strains. It serves also as the basis for the panmetabolome and panphenome by integrating curation with the analysis of gain and loss, and construction of species-specific metabolic networks linking the annotations to observed phenotypes.

Abstract #15 Serology study of Q-fever in cattle and small ruminants in Georgia E. Mamisashvili1*, M. Donduashvili1, K. Goginashvili1, T. Tigilauri1, L. Gelashvili1, O. Parkadze2, L. Avaliani2, L. Ninidze2, I. Menteshashvili2, M.J. Stuckey3, B.B. Chomel3 1Laboratory of the Ministry of Agriculture, Tbilisi, Georgia 2National Food Agency, Tbilisi, Georgia 3Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA,

Introduction: Coxiella burnetii (Family of Coxiellaceae) is a zoonotic pathogen that can infect many different mammalian species. In this study, we surveyed twelve regions of Georgia for C. burnetii infection in cattle and small ruminants. The last known cases of human and animal Q-fever in Georgia were recorded in 1955 when a total of 17 strains were isolated (seven strains from human samples and 10 from domestic animal samples). Materials and Methods: Samples were collected from nine regions of Georgia by teams from the National Food Agency (NFA) of the Ministry of Agriculture (MoA). Blood, serum, milk, and smear samples were taken from cattle, sheep, and goats. In total, 2,230 blood samples were collected from these animals, serum was tested by serological methods (IFA and ELISA). All samples were tested at the BSL-2 plus level. Results: Samples were tested using IFA (n=1,474), and some were tested using ELISA (n=756). Among samples tested, 183 were from goats, 799 from sheep, and 1,248 from cattle. Out of 183 goat samples, eight (4.4%) were ELISA positive and three suspect, with seven being IFA positive and seven suspect (from the same animals). Out of 799 sheep serum samples, four were ELISA positive and seven (0.87%) were IFA positive. Out of 1,248 cattle serum samples tested, 31 were ELISA positive and six suspect, with three being IFA positive and nine suspect. Conclusions: The ELISA and IFA serology methods described above were used to detect antibodies in serum. ELISA testing did not demonstrate the ability to differentiate phase I and phase II Coxiella infection. Differentiation is possible using IFA, which allows for detection of both the acute and chronic forms of disease.

29 Abstract #16 Complete Tick Mitochondrial Genomes from the Ion Torrent PGM Zachary C. Holmes1, T. Brian Shirey2, Jasmine R. Hensley1, Maria L. Zambrano1, and Gregory A. Dasch1* 1Rickettsial Zoonoses Branch, CDC, Atlanta, GA 30329; 2Enteric Diseases Laboratory Branch, CDC, Atlanta, GA 30329

Complete genomic sequence data for Ixodid (hard) ticks are severely limited in availability. Ixodid genomes are thought to range from 1.04-7.1 Gb and contain large stretches of different families of heteropolymeric DNA, making them a challenge even to modern Next Generation Sequencing (NGS) and assembly methods. Mitochondrial sequences are important targets for population genetics studies and taxonomic identification but robust databases for ticks are available for only four gene fragments. We have previously assembled complete mitochondrial genome sequences for Dermacentor variabilis and for Amblyomma americanum from ultrahigh throughput Illumina HiSeq metagenomics data sets. However, this approach is not an efficient general strategy nor is it cost effective if only mitochondrial data is needed. Consequently, we have evaluated direct sequencing of enzymatically sheared shotgun DNA libraries on the Ion Torrent PGM (318v2 chips with 200 or 400 bp chemistry) to determine whether alcohol preserved tick samples could serve as suitable sources of mitochondrial DNA (Qiagen spin column extracted) for full mitochondrial assemblies. Sequence read depth and quality with 400 bp chemistry was greatly inferior to those obtained with the 200 bp chemistry. Raw and quality trimmed sequences equences were mapped with Geneious to the HiSeq A. americanum and D. variabilis assemblies, and the complete A. triguttatum, A. fimbriatum, A. cajennense, D. nitens and D. silvarum mitochondrial sequences from GenBank. The read map sequences and full sequences obtained were also assembled de novo in Geneious and CLC Genomics Workbench, respectively. Ion Torrent PGM read coverage was variable between samples which limited heteroplasmy analysis. The duplicate control regions and other gaps were verified with Sanger sequencing. The assembled sequences were initially annotated using the MITOS WebServer and translations of open reading frames checked with Geneious. Gene order, mitochondrial size (14-15 Kb, number of tRNAs (21-22) and rRNAs (2), and number of proteins (13) were those expected for Amblyomma and Dermacentor and consistent with mitochondria of other hard ticks. Mitochondrial sequences were obtained for Amblyomma maculatum (GA, OK), D. occidentalis, D. andersoni, and for samples of D. variabilis from Georgia, Ohio, and Virginia. Because of the limited sequence read depth on the Ion Torrent PGM and the requirement for some Sanger sequencing for assembly confirmation, longer read methodologies using PacBio RSII mitochondrial amplicons or multiplexed barcoded amplicon sequencing on the Illumina NextSeq or MiSeq may be preferable where heteroplasmy analysis is of interest. However, the Ion Torrent data obtained here suggests that the heteroplasmy is minimal for the tick species that were examined.

Abstract #17 First report of Theileria cervi infection in Roe deer (Capreolus capreolus) in the Republic of Korea Kyoung-Seong Choi1,*, Yu-Jung Han1, Joon-Seok Chae2, Do-Hyeon Yu3, Jinho Park4, Bae-Keun Park5, Hyeong-Chul Kim6 1College of Ecology and Environmental Science, Kyungpook National University, Sangju 37224, Republic of Korea; 2Laboratory of Veterinary Internal Medicine, BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea; 3College of Veterinary Medicine, Chonnam National University, Gwangju 61186, Republic of Korea; 4College of Veterinary Medicine, Chonbuk National University, Iksan 54596, Republic of Korea; 5College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea; 6College of Veterinary Medicine, Kangwon National University, Chuncheon 24341, Republic of Korea

Objective: The genus Theileria species can be classified into two main groups depending on their targets (lymphocytes or erythrocytes). T. annulata and T. parva are the causative agents of lymphoproliferative disease with high mortality and morbidity, and are commonly distributed in tropical regions. T. buffeli/orientalis causes benign theileriosis, with reports of mild or asymptomatic disease in East Asia. In the ROK, Theileria sp. has been reported in several wild animal species, such as Chinese water deer and Korean water deer, while infection with other Theileria species has not been observed in wild animals. Materials and methods: Blood samples were collected in 2015 from a total of 34 animals found at 6 different Wildlife Rescue Center. These included 18 Korean water deer (Hydropotes inermis argyropus), 15 Korean raccoon dogs (Nyctereutes procyonoides koreensis), and one roe deer (Capreolus capreolus). The AccuPower Theileria and Rickettsiales

30 3-Plex (Anaplasma, Ehrlichia, and Rickettsia) PCR Kit were used. For nested PCR amplification of Theileria spp., the V4 hypervariable region of the 18S rRNA gene was used. Results: The isolate found in roe deer was identified as T. cervi and diverged from the other Theileria isolates. Based on phylogenetic analysis, the T. cervi isolate (KU565349) obtained in this study was clustered with a deer isolate from China (HQ184410; 99.5% identity) and an elk isolate from the USA and Canada (U97054, Type F; 98% similarity). According to the V4 hypervariable region of the 18S rRNA, the T. cervi isolate belongs to a type F, which separates it from type G and other bovine theileriosis isolates. Conclusion: This study is the first to report T. cervi infection in the roe deer. As our isolate is most similar to another isolate from China, it appears that ticks introduced from China can spread among wild animals, which is an association with geographical location. Although the tick vector involved in the transmission of T. cervi was not identified in this study, we cannot rule out the possibility that ticks capable of transmitting T. cervi are present in the ROK.Acknowledgements: This work was carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ010092), Rural Development Administration, Republic of Korea.

Abstract #18 Evaluation of Gulf Coast ticks (Acari: Ixodidae) for Ehrlichia and Anaplasma species Michelle E.J. Allerdice*, Joy A. Hecht, Sandor E. Karpathy, Christopher D. Paddock Rickettsial Zoonoses Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention 1600 Clifton Rd NE, Mailstop G-13, Atlanta, GA 30329

Amblyomma maculatum (the Gulf Coast tick) is an aggressive, human-biting ixodid tick distributed throughout much of the southeastern United States and is the primary vector for Rickettsia parkeri, an emerging human pathogen. A. maculatum have diverse host preferences that include white-tailed deer, a known reservoir for many pathogenic Ehrlichia and Anaplasma species. To examine more closely the potential role of A. maculatum in the maintenance of various pathogenic Ehrlichia and Anaplasma species, we examined DNA samples of 470 questing adult A. maculatum collected from 6 U.S. states by using broad-range Anaplasmataceae and Ehrlichia and Anaplasma genus-specific PCR assays. Of the samples tested, 4 (0.9%) were positive for DNA of Ehrlichia ewingii, 1 (0.2%) was positive for Anaplasma platys, and 1 (0.2%) was positive for a previously unreported bacteria most closely related to an uncultivated Ehrlichia species from Haemaphysalis longicornis ticks in Japan. No ticks contained DNA of Ehrlichia chaffeensis, Ehrlichia canis, the Panola Mountain Ehrlichia, or Anaplasma phagocytophilum. This is the first identification of E. ewingii and A. platys in questing Gulf Coast ticks; nonetheless the low prevalence of these agents suggests that A. maculatum is not likely an important vector of these zoonotic pathogens.

Abstract #19 Knowledge of rickettsiosis among first contact physicians from a rural community in Mexico Ranjan Premaratna1, César Lugo-Caballero1*, Karla R. Dzul-Rosado1, Irving Dzul-Tut2, Angel Balam-May2, Juan José Arias- León2and Jorge Zavala-Castro1 1Regional Research Center “Dr. Hideyo Noguchi,” Autonomous University of Yucatán, Mexico 2 Faculty of Medicine, Autonomous University of Yucatan, Mexico

The ecological conditions of Yucatan made it a suitable region for the acquisition of vector borne diseases such as rickettsiosis. As the epidemiological burden of this disease shows an alarming increase of severe cases, the early establishment of diagnosis and therapeutics by first contact physicians is a critical step that is not being fulfilled due to several reasons, including poor knowledge. The objective of this work was to determine the level of knowledge related rickettsiosis among rural first contact physicians of Yucatan. We applied a survey was applied to 90 first contact physicians from rural clinics of Yucatan, which included 32 items related to the diagnosis, treatment and prevention of rickettsiosis. Answers were analyzed by central tendency statistics. Strikingly, only 54% knew the clinic characteristics of the disease, 35% about its diagnostic and 42% the treatment guidelines. Results related to other areas were also under acceptable values for an endemic zone of rickettsiosis. This data supports the idea than a strong campaign for a continuous diffusion of knowledge regarding this disease is needed. In regions with high prevalence of this disease like Yucatan, the impact of these results on its epidemiological burden must be evaluated.

31 Abstract #20 Clinical case: Fatal rickettsiosis in southern Mexico César Lugo-Caballero1*, Karla R. Dzul-Rosado1, Georgina Rodríguez-Moreno2, Karina López-Avila1, Raúl Tello-Martín1 and Jorge Zavala-Castro1 1Regional Research Center “Dr. Hideyo Noguchi,” Autonomous University of Yucatán, Mexico 2 National Medical Center “Ignacio García Téllez”, Mexican Institute of Public Health

Rocky mountain spotted fever (RMSF) is a tick-transmitted disease, caused by Rickettsia rickettsii, which is an intracellular bacteria. Mexico has the ecological and socioeconomic characteristics suitable for the spread of this disease however, due to its clinical similarity with other exanthematic febrile diseases like Dengue or Chikungunya, its diagnosis is often delayed. As a consequence, the treatment is not early established which has severe or even fatal consequences. The objective of this work is to describe a severe clinical case of RMSF as this is not common especially among toddlers in our region, for its medical diffusion. A female of 2 years old without related background sporadically showed intermittent fever alongside with exanthema and abdominal pain therefore, was taken to the hospital. There she showed generalized epilepsy, which required assisted ventilation and sedation. Patient showed a rapid evolution with septic shock, low cardiac output syndrome and multi-organic failure. After some hours of its hospital admittance she was diagnosed with acute RMSF by serologic and molecular tests. However, the patient died some hours after this diagnostic. It is important to share this data to the medical community to reduce the proportion of cases with delayed diagnosis and treatment.

Abstract #21 Production and evaluation of candidates for the development of DNA vaccines against R. rickettsii. Karla R. Dzul-Rosado*1, Cesar Lugo-Caballero1, Javier Balam-Romero1 and Jorge Zavala-Castro1. 1Regional Research Center “Dr. Hideyo Noguchi,” Autonomous University of Yucatán, Mexico

In Mexico and Latin America, rickettsiosis is a health problem frequently aggravated by a delay on its diagnosis and treatment, which sometimes results in the death of the patient. Many studies involving the development of vaccines against rickettsiosis have demonstrated that outer membrane proteins could be promising candidates. The objective of this work was to evaluate the immunostimulatory effect of three rickettsial DNA plasmids expressing different regions of rOmpA and rOmpB from R. rickettsii (rOmpA-49, rOmpB-15, rOmpB-24), as potential candidates for DNA vaccines. For this, liposomes containing the plasmids were transfected in macrophages derived from the THP-A cell line by PMA stimulation, and the expression of rickettsial peptides was demonstrated by RT-PCR and IFA. Lymphocytes obtained by gradient centrifugation from patients previously diagnosed and treated for rickettsiosis along with healthy subjects, were grown in co-culture with the transfected macrophages. Lymphoproliferation index was determined by the measure of cytokines in the supernatant along with the reduction of MTT to formazan salts (ELISA). Significative differences were obtained in the proliferation of lymphocytes from patients using the plasmids, being rOmpb-24 the candidate with the most notorious response. We concluded that these plasmids produce an immunostimulatory effect on mononuclear cells, which could result in a response by HLA I, which is desirable to overcome this infection.

Abstract #22 An ecological analysis of the bacterial microbiome of Dermacentor andersoni in populations with differing vector competence Cory A. Gall*1, 2, Glen A. Scoles3, Krisztian Magori4, and Kelly A. Brayton1, 2,4 1Department of Veterinary Microbiology and Pathology, Washington State University; 2Department of Tropical Veterinary Diseases, University of Pretoria, Pretoria, South Africa; 3USDA-ARS, Animal Disease Research Unit, Pullman, WA; 4Department of Biology, Eastern Washington University; 5School for Global Animal Health, Washington State University

Ticks are obligate ecoparasitic arthropods that feed on terrestrial vertebrates and are of medical-veterinary importance due to their ability to inflict harm to humans and animals. The Rocky Mountain wood tick, Dermacentor andersoni, is a 32 principal vector of bovine anaplasmosis, cased by Anaplasma marginale, which is the most widespread tick-borne pathogen of livestock worldwide. Although ticks are host to pathogenic bacteria, non-pathogenic microbes make up the majority of the microbiome. These endosymbionts have been shown to have a role in fitness, fecundity, and pathogen acquisition. However, only a few studies have analyzed the ecology and behavior of the bacterial microbiome of ticks. The goal of this study was to characterize the bacterial microbiome of D. andersoni ticks examining different ecological factors, focusing on time and space. We have characterized the bacterial microbiome of two populations of D. andersoni ticks with differing vector competence for A. marginale: Burns, Oregon had a high rate (60%) and Lake Como, Montana a low rate (20%). We used Pacific Bioscience SMRT sequencing platform to sequence 16S rDNA in order to examine the microbiome. Our analysis has shown that the bacterial composition was tissue- and spatial-specific; however, generational variation was dependent on geographic location. Furthermore, our study showed that the microbiomes of laboratory-reared populations were not representative of the respective field population. These results have demonstrated that the bacterial microbiome of D. andersoni was dynamic with the ability to undergo significant change. In order to employ microbiome manipulation as a source of pathogen interference, ecological factors and variations need to be fully understood.

Abstract #23 Phylogeography of Orientia tsutsugamushi Amy Fleshman*1, Kristin E. Mullins*2, Jason Sahl1, Crystal M. Hepp1, Daniel Paris3, Sabine Dittrich4, Paul Newton4, Allen L. Richards2,5, Talima R. Pearson1 1Northern Arizona University, Flagstaff, AZ, United States 2Naval Medical Research Center, Silver Spring, MD, United States 3Mahidol-Oxford Tropical Medicine Research Unit, Bangkok, Thailand, 4Wellcome Trust–Mahosot Hospital–Oxford Tropical Medicine Research Collaboration, Vientiane, Lao People's Democratic Republic 5Uniformed Services University of the Health Sciences, Bethesda, MD, United States

Orientia tsutsugamushi, formerly Rickettsia tsutsugamushi, is an obligate intracellular pathogen that causes scrub typhus, an acute febrile disease with high morbidity and diverse clinical presentation. Scrub typhus is transmitted by several species of Leptotrombidium chigger mites and is characterized by a largely irregular geographic distribution. The disease is endemic to regions of Asia, Australia, and islands of the western Pacific, encompassing over 1 billion people, but the distribution is expanding with recent cases reported from United Arab Emirates, Chile, and Africa. Previous work to understand population genetics of O. tsutsugamushi isolates has been based on subgenomic sampling methods, and whole genome characterization has been based on analyses of single genomes. In this study, we compared 40 whole genomes from geographically dispersed areas, allowing for the limited detection of geographic structuring, along with delineation of within clade recombination, but among clade resolution. Here, we present the phylogeographic history of Orientia tsutsugamushi using a Bayesian phylogenetics approach, where we incorporate both location and genomic information to present a model of spatio-temporal diffusion.

Abstract #24 Detection of spotted fever group rickettsiae in ticks from the Republic of Korea and the development of quantitative real-time polymerase chain reaction assays for Rickettsia monacensis and Rickettsia sp. LON-13 Ju Jiang1*, Jonathan Erber1, Allison Dauner1, Theron Gilliland Jr1, Shuenn-Jue Wu1, John S. Lee2, Heung-Chul Kim3, Sung- Tae Chong3, Won-Jong Jang4, Terry A. Klein5, Allen L. Richards1 1Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland, USA 2Biomedical Advanced Research and Development Authority, Dep’t of Health and Human Services, Washington, DC 35th Medical Detachment, 168th Multifunctional Medical Battalion, 65th Medical Brigade, Unit 15247, APO 4Department of Microbiology, College of Medicine, Konkuk University, Seoul, Republic of Korea 5Public Health Command District-Korea, 65th Medical Brigade, Unit 15281, APO AP 96205-5281, USA

Serological and molecular evidence have shown spotted fever group (SFGR) and typhus group (TGR) rickettsia infections existed in febrile patients in and military populations deployed to the Republic of Korea (ROK). During tick surveillance for rickettsial pathogens in places near military installations and training sites, Rickettsia monacensis was found in Ixodes 33 nipponensis collected from rodents in Gyeonggi and Gangwon provinces ROK, with relative high prevalence (44.2%). Another rickettsial isolate, Rickettsia sp. LON-13, was detected in Haemaphysalis longicornis ticks collected from Gyeonggi province in 2004. It was found in all 5 H. longicornis tick pools (60 ticks/pool) assessed and was characterized by multilocus sequence typing (MLST). The sequences of 6 genes (rrs, 17KDa, gltA, ompA, ompB and sca4) showed this molecular isolate to be closely related to Rickettsia japonica and Rickettsia heilongjiangensis (similarity 97.3-99.7%). To better support future rickettsial investigations in the ROK, two quantitative real-time polymerase chain reaction (qPCR) assays were developed targeting specific sequences of ompA and ompB for R. monacensis (Rmonac) and Rickettsia sp. LON-13 (RspHlR), respectively. These assays were optimized and their specificities were confirmed by testing against DNA panels that included 20 species of closely related rickettsiae, 15 near neighbor bacteria, and human and mouse DNA. The limit of detection (LOD) was determined to be 10 and 20 copies for Rmonac and RspHlR, respectively, using serial dilutions of the constructed positive control plasmids. In addition, RspHlR was evaluated by testing a subset of H. longicornis collected from Gyeonggi province in 2015. These agent-specific assays are not only important in rickettsial surveillance studies for the ROK but also for other endemic locations worldwide.

Abstract #25 Proposed study on the prevalence of coxiellosis among livestock in western Azerbaijan M.G. Abdullayev Azerbaijan Veterinary Scientific Research Institute, Baku, Azerbaijan

Q Fever (coxiellosis in animals) is a zoonotic disease caused by the bacterium Coxiella burnetii. The primary animal reservoirs for human infections of Q fever are cattle, sheep and goats, which can shed the pathogen in urine, feces, milk and birth products. Humans usually acquire Q fever by inhalation of C. burnetii aerosolized from contaminated materials originating from infected animals. The impact of this zoonotic infection in animals can be significant, including abortion, fever, decreased livestock production, and economic losses to farms engaged in animal husbandry, in addition to the disease risk to humans who care for livestock. This proposed study will determine the prevalence of coxiellosis in cattle, sheep and goats in the western region of Azerbaijan. C. burnetii infection in humans has occurred historically in Azerbaijan, but current data is very limited. Studies of C. burnetii infection in animals have not been conducted in Azerbaijan. This study will be the first regional characterization of coxiellosis in Azerbaijan. The results from this study will facilitate development of strategies for the future surveillance, control and mitigation of Q fever. The western economical region of Ganja-Gazakh which includes the rayons of Agstafa, Dashkasən, Gadabak, Ganja, Goranboy, Goygol, Qazax, Naftalan, Samux, Shamkir, and Tovuz will be used as the study area. This is a high migration area of Azerbaijan, with increased animal contact as animals move from summer to winter pasture and back throughout the year. Laboratories in this region have also observed a higher number of undiagnosed cases of livestock abortion over the past 5 years. Government veterinary records will be used to estimate the number and locations of cattle, sheep and goats in each of the rayons and a multi- stage sampling plan will be used to proportionately select animals from rayons and farms. Trained regional veterinary officers will collect blood, feces, vaginal and milk samples from cattle, sheep and goats and record information on geographical location for mapping capability. Samples will be tested for C. burnetii in local laboratories with serological methods; confirmatory testing will be performed using molecular methods in the central Baku laboratory. This study will provide the first characterization of coxiellosis in livestock in western Azerbaijan and allow development of epidemiologic maps highlighting the geographic distribution of this pathogen in livestock in the region. The results of this study will enhance public health awareness, and facilitate future development of surveillance, prevention and control strategies for this zoonotic disease throughout the country. This study may also be used as a model for C. burnetti detection in other regions of Azerbaijan in order to develop a clear picture of this pathogen in the country.

Abstract #26 Outbreak Investigation of Scrub Typhus in Thailand: 15 Year Experience Wuttikon Rodkvamtook1, Darunee Utennam1 Narupon Kuttasingkee1, Yutthapong Sudsawat1, Chusak Molito2, Allen L. Richards3,4 Wei-Mei Ching3,4 Chien-Chung Chao3,4 Piyada Linsuwanon5 and Jariyanart Gaywee1 1. Armed Forces Research Institute of Medical Science (AFRIMS), Bangkok 10400, Thailand. 2. Office of Disease prevention and control 12, Songkhla. 3. Viral & Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland, 20910, USA. 34 4. Preventive Medicine & Biometrics Department, Uniformed Services University of the Health Sciences (USUHS), Bethesda, Maryland, USA. 5. U.S. Army Medical Directorate Armed Forces Research Institute of Medical Science, Bangkok, Thailand.

Scrub typhus, a mite-borne disease characterized by high fever, headache, muscle pain, myalgia, skin rash, and eschar lesion. The disease causes by an obligate intracellular Gram-negative bacterium, Orientia tsutsugamushi which its life cycle naturally maintained in chiggers (trombiculid mite larvae) and small mammal. Human become infected via chigger bite. The disease is epidemic in Asian Triangle. This report summarized the results of scrub typhus outbreak investigation in 3 regions of Thailand during the past 15 years. In central Thailand, scrub typhus outbreaks occurred in Royal Thai Army troops during field training in 2002 and 2013. Approximately 30% of soldiers had febrile illnesses and 10% were seropositive to O. tsutsugamushi by IFA. A cluster of 26 seropositive scrub typhus patients was identified in Hill tribe villagers living in Pongyaeng village, Maerim District, Chiangmai Province, northern Thailand during 2006-2007. For the southern Thailand, a case of scrub typhus was reported in tsunami evacuated population in early 2005. Recently, 170 patients with scrub typhus-like clinical symptoms were reported in 5 districts of Patthalung Province during December 2015. Forty-four percents of 36 selected patients in Paphayom district were IFA positive for O. tsutsugamushi. Survey of reservoir hosts and vectors were performed in outbreak areas to investigate for the disease transmission cycle. The results indicated these areas were at risk for scrub typhus by the presence of infected rodents which infested with well-described scrub typhus vectors, Leptotrombidium sp. chiggers. Specific information regarding scrub typhus transmission in endemic areas is crucial to establish an effective disease prevention program. The use of insect repellant, preventive concern, health education and disease surveillance are strongly enforced during Army training in risk areas and proved effectiveness. Concern to prevent scrub typhus was also raised in civilian living in risk areas and public health authorities as a results of outbreak response.

Abstract #27 EcxR is a global gene regular in Ehrlichia chaffeensis Laxmi U. M. Jakkula*, Huitao Liu, Ying Wang and Roman Ganta Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen has a biphasic developmental cycle when it infects vertebrate monocytes/macrophages and tick cells. Host-specific differences in the gene expression from many genes of E. chaffeensis are also reported. It is entirely unknown how the organism regulates its gene expression during its developmental cycle and for it adaptation to vertebrate and tick hosts. We recently mapped promoters of several genes of the pathogen recognized by its only two sigma factors; σ32 and σ70. Much remains to be understood about the regulation of gene expression. In the current study, we tested the hypothesis that one or more of the pathogen transcription regulators play an important role in regulating the gene expression. In support of testing this hypothesis, we initiated the characterization of five putative transcription regulators of the pathogen (CtrA, EcxR, HU, MerR and Tr1) for their roles in influencing the promoter activities of 7 genes encoding for ClpB, DnaK, GroES/L, Hup, p28-Omp 14, p28-Omp 19, and Tr1. Our studies involved in preparing recombinant transcription regulators and their interactions with the promoter regions of the above listed 7 genes. Electrophoretic mobility shift assays (EMSAs) were performed with Tr1, CtrA and EcxR for the 7 promoter segments, and specific interactions were observed for all of the promoters with EcxR. Specific binding of the EcxR was verified by South-Western blot analysis for the promoters of ClpB, DnaK, GroeS/L, p28-Omp 14 and 19, and Tr1. We then investigated then impact of EcxR binding on the promoter activities of ClpB, DnaK, GroeS/L p28-Omp 14 and 19; the EcxR binding resulted in the variable levels of transcription enhancement from 20-60%. Enhancement of the promoter activity was validated independently for p28-Omp 14 and 19 gene promoters in the E. coli surrogate system where E. chaffeensis σ70 is complemented in the absence of E. coli σ70. Finally, we confirmed specific binding of the EcxR to the Tr1, p28-Omp 14 and 19 gene promoters by yeast one-hybrid assay. This is the first detailed study focused on evaluating the contributions of several transcription regulators and our studies demonstrate that the EcxR is a global regulator that specifically binds and enhances the promoter activities of several E. chaffeensis genes. (This work was supported by the PHS grant # AI070908 from the National Institute of Allergy and Infectious Diseases, USA. This work also received support from the CEVBD, Kansas State University.) 35 Abstract #28 Regulation of pulmonary long non-coding RNAs in a murine model of Rocky Mountain spotted fever Imran H. Chowdhury1, Abha Sahni1, Hema P. Narra1, Kamil Khanipov2, Yuriy Fofanov2, and Sanjeev K. Sahni1 1Department of Pathology and Institute for Human Infections and Immunity, The University of Texas Medical Branch Galveston, TX 2Department of Pharmacology, The University of Texas Medical Branch Galveston, TX

Recent advances in genomics have revealed that non-coding RNAs constitute a majority (~98%) of the transcriptome. Long non-coding RNAs (lncRNAs) include a diverse class of transcribed RNAs of ˃ 200 nucleotides that do not encode proteins, are classified into different subtypes, and have important regulatory functions in pathways of inflammation and immunity. To investigate their roles in the host response to Rickettsia infection, we performed RNA-sequencing on the lungs of mock- and R. conorii-infected C3H/HeN mice. The resultant reads mapped to a total of 74,964 ncRNAs, of which 206 and 277 ncRNAs were significantly up- and down-regulated, respectively. After filtering out 27 short ncRNAs, we mapped the genomic sequence of the remaining 179 up-regulated lncRNAs to the ChIP-seq data of mouse lung epigenetic markers H3K4Me1 and H3K4Me3 for cataloging enhancer and can-lncRNA/promoter associated lncRNAs, respectively. This enabled removal of the transcripts overlapping with or sharing transcription start sites with coding genes and identification of 27 up-regulated elncRNAs for further mapping against mouse lung ChIP-seq data for POLR2A, p300, CTCF, and DNase hypersensitivity sites to differentiate into active and poised transcripts. Finally, three elncRNAs [NONMMUT013718, NONMMUT024102, and NONMMUT003295] were confirmed for significantly higher expression in infected mouse lungs by qRT-PCR. ChIA-PET and Hi-C sequencing data further revealed that NONMMUT013718 and NONMMUT024102 elncRNAs interact with proximal coding genes ID2 and Apol10b and accordingly, higher expression of both of these coding genes at the transcriptional level was also confirmed via qRT-PCR. To conclude, combinatorial application of RNA-seq and systematic downstream analyses has, so far, allowed us to identify two regulatory elncRNAs with increased expression and to further confirm up-regulation of their cognate target genes involved in T-cell polarization and lipid transport mechanisms. Exploration of elncRNA-mediated innate/adaptive immune regulation during host interactions with pathogenic rickettsiae merits further attention and is therefore ongoing.

Abstract #29 Neither too much nor much less: Balancing act of a promising drug target in Rickettsia conorii Jignesh G. Patel*#, Hema P. Narra#, Krishna Mohan, Aishwarya Sahni, Amber Singh, Casey L.C. Schroeder, Abha Sahni, and Sanjeev K. Sahni Department of Pathology, The University of Texas Medical Branch, Galveston, TX

Reductive evolution resulting from gene loss/decay is now appreciated as one of the major driving forces shaping the genomes of obligate intracellular bacteria. Rickettsia species have independently lost several genes encoding de novo biosynthetic pathways, nucleotide biosynthesis, and energy metabolism during the course of evolution. However, a core set of genes implicated in virulence and survival have been retained in most rickettsial genomes. We have identified RC0497, a putative N-acteylmuramoyl L-alanine amidase, in R. conorii to be constitutively expressed at both 3 and 24 h post-infection of Human Microvascular Endothelial Cells (HMECs) in vitro. The homologs of RC0497 are characterized as peptidoglycan hydrolases required for cleavage of the bond between N-acetylmuramoyl and L-alanine residues in the cell wall glycoproteins, and are implicated in cell separation in several other bacteria. BLASTp analysis revealed that RC0497 homologs are highly conserved in rickettsial species (excluding typhus group) and other members belonging to α- proteobacteria. By quantitative RT-PCR, we have further confirmed that RC0497 is significantly upregulated at very early stages of infection (15-30 minutes), and then stably expressed throughout the course of R. conorii infection of HMECs in vitro. We have cloned, expressed, purified, and performed circular dichroism spectroscopy of RC0497 to determine the protein secondary structure. The protein predominantly exhibited an α-helix structure, and the presence of zinc as a cofactor resulted in a conformational change. Confocal microscopy on E. coli expressing R. conorii RC0497 revealed that the protein is predominantly localized to the cell wall and intriguingly, into the septal regions of replicating bacteria. Furthermore, increased expression of RC0497 resulted in elongated, vacuolated and potentially damaged cell structures.

36 Further studies are in progress to functionally characterize the role of RC0497 in rickettsial multiplication and pathogenesis, and as a possible therapeutic/vaccine strategy.

Abstract #30 Biomarkers for Amblyomma maculatum-tissues and associated rickettsiae John V. Stokes1*, Gail M. Moraru1, Mariola J. Edelmann2, and Andrea S. Varela-Stokes1 1College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi 2College of Agricultural and Life Sciences, University of Florida, Gainesville, FL

Rickettsial infection in tick tissues can be determined using various approaches, including molecular assays and microscopy. However, the use of tick tissue-specific biomarkers would allow for more specific and potentially more rapid rickettsial detection within individual tissues of infected ticks. While testing the utility of flow cytometry on discriminating midgut and salivary gland tissues from cell suspensions using whole Amblyomma maculatum, we turned to proteomics to identify differentially expressed proteins in these tissues. Using LC-MS/MS (LTQ Velos Orbitrap mass spectrometer) analysis of midgut and salivary gland tissues from female A. maculatum, we identified over 800 cytosolic proteins based on A. maculatum and I. scapularis databases in the National Center for Biotechnology Information (NCBI) and UniProt. Of these, over 500 were differentially expressed in either tissue (based on Fisher’s exact test with false discovery rate of 0.1% and protein probability of 99%). Proteomics analysis led to identification of two potential biomarkers: phosphomannomutase in salivary glands and isocitrate dehydrogenase in midgut, with 37-fold and 14-fold differences in abundance, respectively. Selected peptides were produced and conjugated to KLH, then submitted for monoclonal antibody production. Fusions have been performed and we are currently awaiting supernatants for initial screening of hybridomas. Resultant antibodies that show minimal or no cross-reactivity will be used for microscopy of fixed (permeabilized) cells in suspension or in situ to support cell types in with rickettsiae may be found. Currently, we are performing transcriptomics on A. maculatum midgut and salivary gland tissues to confirm or further inform our proteomics data. We anticipate that the identification of A. maculatum-specific biomarkers, in conjunction with biomarkers for associated rickettsiae, will aid in understanding rickettsial interactions with the tick as well as rickettsial maintenance and transmission.

Abstract #31 Gene expression profiles of the de novo folate biosynthetic pathway from a Rickettsia Endosymbiont in vivo, in Ixodes pacificus, and in vitro, in the Ixodes scapularis ISE6 tick cell line Kristine Teague*, Maryam Alowaysi, Junyan Chen, and Jianmin Zhong Department of Biological Science, Humboldt State University, Arcata, CA, 95521

The nature of symbiotic relationships between ticks and their endosymbionts is unclear. There is however, evidence suggesting a nutritional component and one such nutrient, folate, plays a fundamental role in metabolism and biosynthesis of nucleotides, and amino acids. We have identified the complete folate biosynthetic pathway (folA, folC, folE, folKP, ptpS) in Rickettsia species phylotype G021, the endosymbiont of Ixodes pacificus. We hypothesize that environmental temperature would affect folate gene expression in infected ISE6 cells and that genes would be upregulated in I. pacificus, following a blood meal, to accommodate the physiological changes associated with feeding, molting, and oviposition. We analyzed the expression of folA, folE and folKP in the infected ISE6 cells at 25°C and 36°C as well as flat and engorged I. pacificus. Total RNA and DNA were extracted from ISE6 cells, flat and engorged I. pacificus, and gene expression quantified for each folate gene using RT-qPCR. Rickettsial methionyl-tRNA ligase was used to normalize the expression of each gene and the average folA/tRNA ligase ratio at 25°C and 36°C was 912.5±737.6 and 396.2±139.6, respectively. The average ratios for folE/tRNA ligase and folKP/tRNA ligase at 25°C and 36°C were 55.8±16.3 (25°C) and 39.8±7.64 (36°C), and 3.38±0.39 (25°C) and 3.52±1.14 (36°C), respectively. A t-test showed no significant difference in gene expression between 25°C and 36°C with p-values of 0.11, 0.055, and 0.78 for folA, folE, and folKP, respectively. Additionally, preliminary data with adult I. pacificus samples, suggest different results, with an average folE/tRNA ligase ratio of 4.36±0.44 and 507.08±496.41 for flat and engorged ticks, respectively. These data suggest that folate genes are expressed by Rickettsia species phylotype G021 and may provide evidence for a nutritional symbiosis between phylotype G021 and I. pacificus. 37 Abstract #33 Spotted Fever Group Rickettsia Adaptation to the Mammalian Host Sean P Riley, Ludovic Pruneau, Juan J Martinez Vector-Borne Disease Laboratories, Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, Louisiana 70803

All organisms, from bacteria to humans, need to “recognize” where they are, and respond appropriately. Pathogens like the spotted fever group (SFG) Rickettsia species must sense their location in the infectious cycle, and produce factors necessary for survival within that specific host. This is likely to be particularly important for vector-borne pathogens such as SFG Rickettsia, which must efficiently infect both arthropod and mammal hosts. As such, we have tested the hypothesis that Rickettsia species execute in vivo proteomic changes that are essential to mammalian pathogenesis. We have employed two systems approaches to identify the transcriptional and translational changes elaborated by SFG Rickettsia during mammalian infection as compared to cultured bacteria. Deep sequencing of cDNA libraries (RNA-Seq) was used to profile transcriptional changes occurring within the infected animal. Additionally, cell sorting was utilized to isolate Rickettsia from the infected mammal for iTRAQ analysis define proteomic changes associated with infection. By analyzing both transcriptional and proteomic changes, we were able to more accurately define and validate the adaptations utilized by the bacteria during infection. Future analyses of rickettsial transcripts and proteins specifically expressed in vivo will have the highest potential to yield valuable data pertaining to Rickettsia pathogenesis, and these genes will become the primary candidates for therapeutic intervention and/or genetic manipulation.

Abstract #34 The Coxiella burnetii effector Cig57 hijacks FCHO-mediated vesicular trafficking Eleanor A Latomanski*, Patrice Newton, Hayley J Newton Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia

Coxiella burnetii is an intracellular bacterial pathogen and the causative agent of the zoonosis Q fever. To cause disease, Coxiella establishes a unique intracellular replicative niche by remodelling lysosomes to create the Coxiella-containing vacuole (CCV). The capacity for intracellular replication, and therefore virulence, depends upon the activity of the Dot/Icm type IV secretion system, which translocates approximately 150 effector proteins into the host cell. The function of many of these effectors remains elusive, with a subset now identified that are important for efficient intracellular replication of Coxiella. A transposon mutagenesis screen identified the Dot/Icm effector Cig57 as being essential for intracellular replication of Coxiella. A yeast-two hybrid screen identified the host protein FCHO2 as a binding partner of Cig57. FCHO2 is important for clathrin-coated vesicle formation in the host. It arrives at the plasma membrane early in endocytosis and aids membrane curvature. siRNA silencing and gene knockouts have validated the importance of FCHO1 and FCHO2 for normal intracellular replication of Coxiella and CCV formation. Important endocytic sorting motifs, recognised by adaptor complexes during clathrin-mediated endocytosis, are found in Cig57, and are also important for full function of Cig57 during infection. The presence of Cig57, and more broadly, Coxiella infection, reduces the cells normal capacity for endocytosis via the clathrin pathway. This suggests a role for Cig57 in commandeering this process, potentially in concert with other effectors. We demonstrate that by manipulation of FCHO proteins, Cig57 has a role in exploiting clathrin- mediated vesicular trafficking of the host. We suggest that Coxiella remodels the CCV by using Cig57 to sequester clathrin- coated vesicles. Further work will reveal the mechanism of action of Cig57, to understand why the clathrin-mediated pathway is important for intracellular replication and to cause disease

Abstract #35 Analysis of the Caenorhabditis elegans innate immune response to Coxiella burnetii James M. Battisti, Lance Watson, Myo Naung, Adam Drobish, Ekaterina Voronina and Michael F. Minnick Division of Biological Sciences, University of Montana, Missoula, Montana

Background- Coxiella burnetii is a select agent and an obligate intracellular bacterium that causes a flu-like syndrome in humans (Q fever), as well as stillbirths and abortions in domesticated livestock that commonly serve as reservoirs of this 38 zoonotic agent. Caenorhabditis elegans is a free-living soil nematode and user-friendly model system for studying pathogenic bacteria. Our previous studies demonstrated that infection of C. elegans by Coxiella results in a number of interesting pathological consequences, including a decreased lifespan, abdominal distension, deformed anal region (DAR), and extra-luminal blebs (ELB’s); consistently exhibited by C. elegans following ingestion of live C. burnetii but not live E. coli or dead C. burnetii. In this report, we: 1) attempted to identify Coxiella factors responsible for induction of these C. elegans pathologies and 2) examined pathological consequences of infection in hyper-immune (daf-2) and immunodeficient (sek-1) wormstrains. Methodology/Principal Findings- First, we identified a heat-labile Coxiella factor(s) that participates in generation of C. elegans pathologies. C. elegans fed a diet of autoclave-killed C. burnetii did not exhibit pathologies, whereas a diet of “pasteurization-killed” (72˚C, 15 min) bacteria still generated DAR. Second, compared to wild-type C. elegans, hyper-immune (daf-2) worms showed significantly reduced pathologies (DAR and abdominal swelling) and chose to crawl away from live C. burnetii and starve to death. Conclusions/Significance- These data indicate that an autoclave-labile C. burnetii factor(s) that is not inactivated by pasteurization contributes to the generation of DAR and associated pathologies in infected C. elegans. Further characterization of this factor(s) should lead to a better understanding of the innate immune response. The pathogen avoidance behavior of daf-2 worms suggests that other host-pathogen interactions are involved in the innate immune response of C. elegans to C. burnetii.

Abstract #36 Identifying Novel Host Cell Target Proteins of Bartonella Fic Domains Vaughan Trounson* and Joanna Mackichan School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand

Bartonella henselae is an emerging bacterial pathogen that utilizes an unusual infection strategy; parasitizing the red blood cells of its mammalian host, the cat. B. henselae is transmitted between cats by the cat flea, though occasionally infects humans via cat scratches or bites causing cat-scratch disease (CSD). CSD is characterized by enlarged tender lymph nodes and fever. B. henselae also infects the endothelial cells of both its hosts, likely a factor in disease progression. The bacteria secrete a range of Bartonella effector proteins (Beps) via a Type IV secretion system, directly into endothelial cells, subverting host cell processes and resulting in internalization in the cell. Beps have a range of functions, many of which are not fully characterized. B. henselae secretes three Beps (BepsA-C) that contain a filamentation induced by cAMP (Fic) domain and a Bartonella Intracellular Delivery (BID) domain, with BepA being the best studied. BepA’s BID domain is responsible for intracellular delivery as well as inhibition of apoptosis by the host cell, however the exact function of the Fic domain remains unknown. Fic-containing bacterial toxins catalyse the transfer of an AMP moiety from ATP onto a host cell protein. The host protein is usually a central signalling molecule such as members of the Rho GTPase protein family. Adenylylation frequently inactivates these proteins, resulting in disrupted host cell processes and cytotoxicity. BepA has previously been shown to possess adenylylation activity, although the host target protein(s) are unknown. Evidence suggests that these are novel targets and not members of the Rho GTPase family. This project aims to determine the identity of the host target protein(s) that are adenylylated by the Fic domain of BepA, using a click chemistry based approach and purified Bep-Fic proteins incubated with whole cell extract. These studies will yield new insights into the interactions between B. henselae and host endothelial cells, which may lead to new medical interventions against this pathogen and related species.

Abstract #37 Bacteria Transmitted by Ixodes scapularis Ticks Elicit a Novel Immune Deficiency Circuit Dana K. Shaw1*, Erin E. McClure1, Lindsey J. Brown2, Lei Liu3, Kathleen DePonte3, Alexis A. Smith4, Utpal Pal4, Erol Fikrig3,5, Joao H.F. Pedra1 1Department of Microbiology and Immunology, University of Maryland School of Medicine; 2Inst. of Human Virology, Dep’ts of Medicine and Microbiology and Immunology, Univ. of Maryland School of Medicine; 3Section of Infectious Diseases, Department of Internal Medicine, Yale University; 4Dep’t of Veterinary Medicine, Virginia-Maryland Reg. College of Veterinary Medicine, Univ. of Maryland, College Park; 5Howard Hughes Medical Institute, Chevy Chase, MD 20815

39 Tick-borne diseases are the most common arthropod-borne illnesses in the Northern hemisphere. Despite their public health relevance, it remains mostly unknown how microbial pathogens persist within the tick vector and how the immune system responds to infection. Here, we reveal a novel molecular circuit where the E3 ubiquitin ligase x-linked inhibitor of apoptosis protein (XIAP) interfaces with the Immune Deficiency (IMD) pathway in the tick Ixodes scapularis. The IMD pathway is functionally analogous to the mammalian tumor necrosis factor (TNF) receptor network, and plays a critical role in arthropod humoral responses. Targeted gene silencing of the heterodimeric complex, Bendless/Uev1a, and the positive regulator of the IMD pathway, Relish, caused increased I. scapularis colonization by Borrelia burgdorferi and Anaplasma phagocytophilum, the causative agents of Lyme disease and Human Granulocytic Anaplasmosis. Conversely, overactivation of the IMD pathway by ablating the expression of the negative regulator, Caspar, decreased pathogen infection of ticks. Importantly, bacterial lipid agonists stimulated the I. scapularis IMD pathway, whereas immune priming protected against bacterial infection. Altogether, these findings indicate that I. scapularis relays the immune signal through a distinct modality when compared to insects.

Abstract #38 Experimental evaluation of Peromyscus leucopus as a reservoir host of the Ehrlichia muris-like agent Geoffrey E. Lynn, Jonathan D. Oliver, and Ulrike G. Munderloh Department of Entomology, University of Minnesota, Saint Paul, MN 55108, USA

The Ehrlichia muris-like agent (EMLA) is a newly recognized human pathogen that has been identified in patients in Minnesota and Wisconsin with clinical presentation similar to human anaplasmosis. Field studies have found EMLA in blacklegged ticks (Ixodes scapularis) collected from areas where patients were likely to have been exposed, and laboratory tests have shown that the bacteria can be maintained in a cycle between lab mice and I. scapularis. The reservoir hosts for EMLA are currently unknown, though PCR detection in wild-caught white-footed mice (Peromyscus leucopus) has been reported. Here, we report experimental transmission of EMLA to P. leucopus via feeding nymphs, along with acquisition of the bacteria by co-feeding larvae. Though P. leucopus were able to successfully acquire and transmit EMLA infection, mortality was high (8/10) and period of bacteremia was brief, corroborating the short window of infectivity previously demonstrated in laboratory mouse strains. Potential transmission from infected females to their larval offspring was also evaluated by PCR.

Abstract #39 Tracking Anaplasma phagocytophilum infection in larval Ixodes scapularis Jonathan Oliver*, Geoffrey Lynn, Nicole Burkhardt, Curtis Nelson, Timothy Kurtti, Ulrike Munderloh Department of Entomology, University of Minnesota, St. Paul, MN

Human granulocytic anaplasmosis is the second most reported vector-borne disease in the United States, afflicting several thousand people each year. Despite this, little is known about the relationship between its causative agent, Anaplasma phagocytophilum, and the tick vector of this bacterium, Ixodes scapularis. Within the host tick, intracellular A. phagocytophilum has proven quite elusive to microscopic detection. To better understand the intimate relationship between pathogen and host, we used in situ hybridization microscopy coupled with qPCR to track the progress of A. phagocytophilum HGE2 infection and growth within I. scapularis beginning with larval acquisition of bacteria and proceeding through the life stages of the tick. Ixodes scapularis nymphs are already competent vectors, indicating that salivary gland infection is complete by this stage. The larval transstadial infection/transmission of A. phagocytophilum was examined at weekly intervals throughout molting to develop a higher resolution timeline of the salivary gland infection process. This study provides insight into the tissues involved in early infection and spread of the bacteria within the body of the tick.

Abstract #40 Initial local inflammatory response to Ehrlichia muris-like agent (EMLA) transmitted by ticks Tais B. Saito, Claire Smalley, David H. Walker Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX 40

Ehrlichioses are emerging infectious diseases affecting veterinary hosts and humans. Studies have revealed potential pathogenic mechanisms of infection; however, little is known about the role of the tick vector in establishment of disease. Recently we developed a new mouse model using a human pathogen (E. muris-like agent – EMLA), which mimics several aspects of the natural infection in human patients; and further we established a tick transmission mouse model of EMLA, which allows us to study the effects of the early events on the outcome of the infection. Tick feeding results in formation of a pool of blood and inflammatory cells around the tick mouthparts in the skin, with increased vasodilation mediated by tick saliva. As expected, EMLA transmitted by I. scapularis induced local inflammation in the skin and draining lymph nodes. These early local events induced by the vector tick combined with host inflammatory response to the pathogen can influence how the bacterium is able to establish infection or even how it evades the host response. Using the EMLA tick transmission model, we evaluated the induction of inflammatory cytokines in the bite site of mouse skin and the draining lymph nodes after 24-36 hours of tick attachment. We observed increased levels of expression of genes encoding IL-1 and IL-18 by RT-PCR in skin at the site of EMLA-carrying I. scapularis tick transmission compared to tissues collected from either uninfected tick feeding or from control mice with no ticks feeding. More dramatic changes in IL-18 were observed in the skin compared to IL-1 in mice with EMLA-carrying ticks attached. However, in the draining lymph node the levels of neither cytokine showed significant changes compared to the controls at this time point. Tick feeding itself appeared to have a mild effect on these inflammatory cytokines in skin compared to EMLA tick transmission. These results suggested an inflammatory response induced by Ehrlichia in early transmission by the I. scapularis vector, which is the focus of our studies.

Abstract #41 Ehrlichia chaffeensis TRP120 targets host proteins that differentially influence intracellular survival Tian Luo*, Paige S. Dunphy, and J. W. McBride Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555

Ehrlichia chaffeensis is an obligately intracellular bacterium responsible for the life‐threatening tick transmitted zoonosis, human monocytotropic ehrlichiosis. E. chaffeensis invades and survives in mononuclear phagocytes by modulating cell processes and evading host defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRP) are type 1 secreted effectors involved in functionally diverse interactions with host targets associated with major biological processes of the host cell. In this study, we investigated the influence of 86 TRP120 target proteins on ehrlichial infection by RNA interference. We found that knockdown of 73 (85%) TRP120 target proteins had significant influence on ehrlichial infection by either inhibiting (49% of 86 proteins) or promoting (36%) ehrlichial intracellular survival, including target proteins involved in transcriptional regulation, post‐translational protein modification, cell signaling, intracellular transport, and actin cytoskeleton organization. The inhibition and promotion could occur at different stages of infection. These findings provide more evidences to support the role of TRPs as effectors and reveal that E. chaffeensis exploits tandem repeat proteins to interact with host proteins to promote intracellular survival.

Abstract #42 Pathogen induced host cell ER stress benefits the intracellular bacterium, Orientia tsutsugamushi Kyle G. Rodino*, Lauren VieBrock, Jason A. Carlyon Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA

Perturbation of the endoplasmic reticulum (ER), commonly as a result of ER stress, triggers cellular coping mechanisms tasked with restoring homeostasis, such as the unfolded protein response (UPR). Pro-microbial induction of ER stress by invasive pathogens is an emerging theme in microbial pathogenesis, principally among intracellular organisms. Orientia tsutsugamushi is an obligate intracellular bacterium endemic to the Asia-Pacific region and the causative agent of the potentially fatal human disease, scrub typhus. O. tsutsugamushi encodes a large arsenal of ankyrin repeat-containing proteins (Anks), a historically eukaryotic class of proteins, newly recognized as common among intracellular pathogens and increasingly implicated in the modulation of host cell functions. The majority of these O. tsutsugamushi Anks traffic to the ER when ectopically expressed, a subset of which provoke ER stress, as indicated by the induction of the UPR 41 transcription factors ATF4 and/or XBP1s. ER stress also occurs during O. tsutsugamushi infection, with increases in atf4, xbp1u (un-spliced), and xbp1s (spliced) transcript as observed via qRT-PCR. Given these observations, we investigated the benefit of ER stress to the O. tsutsugamushi lifecycle. In cells treated with the ER stress inducers tunicamycin and thapsagargin, O. tsutsugamushi load was significantly greater compared to vehicle control cells. Conversely, in cells treated with an ER stress inhibitor, tauroursodeoxycholic acid, bacterial load was significantly reduced. These data establish ER stress as an important factor that aids O. tsutsugamushi infection, potentially providing a nutritional benefit, and implicate the Anks as modulators of this stress response.

Abstract #43 Identification of Rickettsia rickettsii Type IV Secretion System effectors Galletti, M.F.B.M.1*; Gaziola, L.M.1; Alexandrino, P. 2; Fujita, A.2; Sauter, I.1; Veliz, M. J. C.1; Voth, D.3; Daffre, S.1; Labruna, M. B.4; Fogaça, A.C.1 1 Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, Brazil; 2 Department of Computer Sciences, Institute of Mathematics and Statistics, University of São Paulo, Brazil; 3 Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock; 4Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine and Animal Science, University of São Paulo, Brazil.

Rickettsia rickettsii is a highly pathogenic intracellular bacterium responsible for the lethality of Rocky Mountain Spotted Fever (RMSF). Previous genetic analysis showed that two environmental stimuli, elevation of temperature and blood feeding, are associated with rickettsia’s virulence reactivation in ticks, but the mechanisms responsible for this phenotype conversion have not been completely elucidated. Studies on the global and tissue-specific transcriptional profile of a virulent R. rickettsii indicated a prominent induction of virulence genes of rickettisiae residing on tick midguts. Most of these genes were components of the type IV secretion systems (T4SS) or potential effector proteins associated with it. In this manner, we performed bioinformatics guided approaches and co-immunoprecipitation to identify the effector proteins associated to the R. rickettsii T4SS. The in silico analyses, performed using three phenotypic different genomes, identified around 34 potential effector candidates in each R. rickettsii strain. In addition, to capture the proteins associated with the T4SS, we produced a VirD4-specific antibody and establish a co-immunoprecipitation assay to validate the effector-VirD4 interactions. In order to identify these proteins, a mass spectrometry analysis of co-immunoprecipitated product is underway. Supported by FAPESP.

Abstract #44 Effect of Rickettsia felis strain variation on infection, transmission, and fitness in the cat flea, Ctenocephalides felis Sean P. Healy1*, Lisa D. Brown1, Melana H. Hagstrom1, Lane D. Foil2 and Kevin R. Macaluso1 1Vector-borne Disease Laboratories, Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 70803; 2Department of Entomology, Louisiana State University, Baton Rouge, LA 70803

Rickettsia felis is a human pathogen transmitted by the cat flea, Ctenocephalides felis as well as an obligate symbiont of the parthenogenetic booklouse Liposcelis bostrychophila. The influence of genetic variability in these two strains of R. felis on host specialization and fitness and possible resulting differences on infection and transmission kinetics in C. felis has yet to be determined. We hypothesize that if there is a genetic element to vector infection, then R. felis strain variation will result in differences in infection, transmission, and fitness in the cat flea. Utilizing an artificial feeding system cat fleas were exposed to a R. felis str. LSU-Lb-infected blood meal and monitored for infection at 7 day intervals for 28 days. Rhodamine B was used to distinguish uninfected recipient and R. felis str. LSU-Lb-infected donor fleas in a horizontal transmission bioassay. Quantitative real-time PCR was used to determine transmission frequency, rickettsial load, and rickettsial infection density in fleas. Dissemination of R. felis str. LSU-Lb was observed on paraffin imbedded sections of infected cat fleas by immunofluorescence assay. The fitness effect of persistent infection of cat fleas with R. felis str. LSU- Lb was determined by total production and female to male ratios in F1 progeny. At 7 days postexposure 76.7% of fleas successfully acquired R. felis str. LSU-Lb. Quantitative real-time PCR determined that both mean R. felis str. LSU-Lb infection load (6.15×106) and infection density (0.76) were significantly greater than R. felis str. LSU infection load 42 (3.09×106) and infection density (0.68) in adult fleas. A persistent R. felis str. LSU-Lb infection was detected at day 28 in adult cat fleas but no infection was found in F1 progeny. Persistent infection of cat fleas with R. felis str. LSU-Lb was not found to alter female to male ratios in F1 progeny. These results demonstrate that R. felis str. LSU-Lb was successfully able to infect cat fleas and did not result in pronounced differences in infection and transmission kinetics compared to R. felis str. LSU. Furthermore, R. felis str. LSU-Lb was not found in F1 progeny and did not disrupt the development and reproductive fitness of infected cat fleas, although it is transovarialy transmitted and contributes to parthenogenisis in the non-hematophagous booklouse L. bostrychophila.

Abstract #45 Immune responses to tick cell-derived versus endothelial culture-derived Ehrlichia muris-like agent (EMLA) in mice Patricia A. Crocquet-Valdes*, Tais B. Saito, and David H. Walker Department of Pathology, Sealy Center for Vaccine Development, WHO Collaborating Center for Tropical Diseases, University of Texas Medical Branch, Galveston TX, 77555-0609.

Human ehrlichioses are tick-transmitted diseases caused by bacteria belonging to the genus Ehrlichia. In 2009, a new strain of ehrlichia closely related to Ehrlichia muris was discovered in patients in the upper Midwestern United States and designated as E. muris-like agent (EMLA). EMLA was also found in Ixodes scapularis ticks in Minnesota and Wisconsin. Phylogenetic analyses performed using the 16S, groEL, dsb and outer membranes protein genes p13 and p29 have shown that EMLA is most closely related to E. muris and may represent a subspecies of E. muris. We have studied the antigens of EMLA to determine the antibodies to which major antigens are present in immune serum. It has recently been shown by our group that the route of transmission in a mouse animal model of EMLA is important in determining the immune response elicited during infection. EMLA is adapted to growth in both vertebrate and invertebrate hosts in its lifecycle. It has previously been shown that macrophage- and tick cell-specific proteins are expressed in E. chaffeensis. We therefore investigated whether different antigens of EMLA would be expressed in different cell lines of both mammalian and tick origin. EMLA was grown in culture in RF/6A cells, an endothelial cell line, and in ISE6, a tick cell line derived from Ixodes scapularis. We performed Western blot analysis using EMLA antigens purified from RF/6A and ISE6 cell lines. We used sera from mice infected with EMLA by intravenous injection (IV), intradermal injection (ID) and through tick transmission. Antibodies against EMLA at day 30 post infection were analyzed by Western immunoblotting. Immunodominant bands representing antibodies against 250-, 155-, 140-, 125-, 87-, 75-, 62-, 28- and 15-kd antigens were identified. Immunogenic proteins from tick cell-derived and endothelial cell-derived cultures were compared using SDS-PAGE. Endothelial- expressed EMLA proteins differed substantially from those expressed in tick cells. More immunodominant bands were seen in the 150- to 75-kd range in the tick cell–derived antigens when compared to the endothelial cell-derived cultures. These data suggest that the host response to EMLA is dependent not only on the route of transmission but also on the host cell of the ehrlichiae.

Abstract #46 Human monoclonal antibodies against tandem repeat and outer membrane proteins inhibit Ehrlichia chaffeensis growth intracellularly Thangam Sudha Velayutham1*, Xiaofeng Zhang1, Gopal Sapparapu2, Gary Winslow3, Nahed Ismail4, James E Crowe2, David H Walker1, and Jere W McBride1 1Department of Pathology, University of Texas Medical Branch, Galveston, TX; 2 Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN; 3Department of Microbiology & Immunology, Upstate Medical University, Syracuse, NY; 4Department of Pathology, University of Pittsburgh, Pittsburgh, PA

Ehrlichia chaffeensis is a gram negative, obligately intracellular pathogen, transmitted by the lone star tick (Amblyomma americanum), that causes the emerging zoonosis human monocytotropic ehrlichiosis. Antibodies are essential for clearance and protection against E. chaffeensis. Although extracellular antibody-mediated immune mechanisms are likely involved, antibody-mediated clearance may also occur intracellularly. Antibodies against major epitopes of known immunoreactive proteins, such as outer membrane proteins (OMP1) and tandem repeat proteins (TRPs), provide protection against E. chaffeensis infection in vivo. Our previous studies demonstrated a reduction in E. chaffeensis infection in cells treated with epitope-specific anti-TRP antibodies, both prophylactically and therapeutically. However, 43 the mechanism whereby antibodies are effective against E. chaffeensis, and the cellular context in which they provide protection is unknown. We investigated the capacity of several E. chaffeensis-specific human monoclonal antibodies (huMAbs), in mediating bacterial clearance in vitro, and the cellular compartment where this inhibition occurs. To identify protective huMAbs, THP-1 cells were treated with antibodies 2 hours prior-, or post-infection, washed extensively and bacterial burden measured 3 days post infection. Anti-TRP32 huMAbs that recognized both the linear and conformational epitopes inhibited E. chaffeensis growth in vitro, but, only the huMAb specific for the linear epitope acted intracellularly. Whereas, an anti-TRP32 huMAb, not specific to either the linear or conformational epitope, was incapable of inhibiting bacterial proliferation. Two anti-OMP-1 huMAbs, also capable of inhibiting ehrlichial proliferation, acted intracellularly. These huMAbs are being further evaluated for the molecular mechanisms involved in intracellular/extracellular antibody- mediated clearance mechanisms in vitro.

Abstract #47 The Effect of Intranasal Infection with “Less Virulent” Spotted Fever Group Rickettsiae on Subsequent Rickettsia rickettsii Pathogenesis in Guinea pigs Edward Shaw1*, Jeff Gruntmier2, and Susan Little2 1Microbiology and Molecular Genetics, 2Veterinary Pathobiology, Oklahoma State University, Stillwater, OK 74078

Rocky Mountain spotted fever (RMSF) is caused by the obligate intracellular bacteria Rickettsia rickettsii and has a 15-25% mortality rate when not treated appropriately. While predominantly acquired from tick bites, it has long been suspected that exposure to aerosolized tick blood/materials containing Rickettsiae could cause RMSF given that respiratory acquired cases have been reported in laboratorians. However, aside from R. rickettsii there is a stunning array of spotted fever group rickettsial (SFGR) that cycle between tick vectors and mammalian hosts in nature. Some of these “less virulent” SFGR are present in tick populations at very high rates and increasingly recognized as real and potential disease agents. However, the extent that they may elicit disease from aerosol exposure and the degree that prior aerosol exposure may affect subsequent R. rickettsii infection is unclear. To investigate this question we intranasally inoculated Guinea pigs with R. parkeri, R. montanensis, R. amblyommii, or sterile PBS and monitored for signs of disease for 14 days. Intranasal inoculation with the SFGR elicited mild fever and no significant reduction in percentage of weight gain relative to PBS controls. In addition, IFA analysis of 21 day sera indicate that significant antibody was generated in all three intranasal infections. Animals were then challenged at 30 days with virulent R. rickettsii Sheila Smith IP and monitored for an additional 14 days. Animals from all three groups experienced mild fever and decreased/delayed weight gain compared to mock challenged PBS controls and all animals survived. The challenged PBS control animals demonstrated profound symptoms and 4/5 were euthanized between days 5 and 6. These data indicate that aerosol exposure to “less virulent” SFGR can cross-protect against subsequent infection by R. rickettsia, a phenomenon that may influence disease dynamics in areas of high SFGR burden.

Abstract #48 Immunopathological Study of Scrub Typhus in Rhesus Macaques Following Intradermal Inoculation of Orientia tsutsugamushi Karp and Gilliam Strains Piyanate Sunyakumthorn1*, Tippawan Anantatat2, Rawiwan Im-erbsin1, Manutsanun Sumonwiriya2, Kesara Chumpolkulwong1, Sirima Wongwairot2, Ajchara Vongsawan2, Susanna J. Dunachie2,5, Matthew D. Wegner1, Christine A Ege1, Allen L. Richards3,4, Nicholas P. J. Day2,5, Daniel H. Paris2,6 1Department of Veterinary Medicine, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; 2Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; 3Department of Viral & Rickettsial Diseases, Naval Medical Research Center, MD, USA; 4Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, MD, USA; 5Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, Oxford University, Oxford, UK; 6Nuffield Department of Clinical Laboratory Sciences, Oxford University, Oxford, UK

Orientia tsutsugamushi, an obligate intracellular Gram-negative bacterium, causes scrub typhus in humans. Emerging evidence from several studies has shown high genetic diversity of O. tsutsugamushi, which may be reflected in differences in the immunopathophysiology of infections caused by different O. tsutsugamushi strains. In this study we compared 44 bacterial dissemination dynamics, clinical manifestations and humoral/cellular immune responses in a rhesus macaque (Macaca mulatta) scrub typhus model using two different O. tsutsugamushi strains: Karp (highly mouse pathogenic strain) and Gilliam (intermediate mouse pathogenic strain). Both strains are highly pathogenic for humans. Previous reports suggest that the Gilliam strain appears to be more virulent and is associated with more severe clinical signs in non-human primates. To assess the virulence of the Gilliam strain, in comparison to the Karp strain, in our Rhesus macaque scrub typhus model two groups of animals (n=4) were intradermally inoculated at both left and right anterior 7.8 thighs with egg-yolk prepared inocula of either Karp or Gilliam strains at 10 muLD50. Blood samples were collected for bacterial quantitation, antibody responses, and immunological bioassays until day 28 post inoculation (pi) when the animals were treated with doxycycline. Macaques infected with O. tsutsugamushi Gilliam strain demonstrated more pronounced eschars and earlier onset of bacteremia (Day 3 pi), while in the Karp strain-infected group bacteremia was detected later on Day 6 pi. Cellular immune responses in the Karp strain-inoculated macaques as determined by ex-vivo IFN-γ ELISPOT were higher than in the Gilliam group on days 12 and 28. At day 80 pi, blood and tissue samples were collected for pathology, long-term immune responses, and immunohistochemistry. Interestingly, the Gilliam-inoculated macaques showed more pronounced cellular immune responses in blood and skin at day 80 pi compared to the Karp group. Long-term memory of cellular immune responses in skin tissues and lymph nodes were detected in both tissues at day 80 pi. Compared to Karp strain, the Gilliam strain caused more severe clinical signs and overall induced more pronounced host immune responses similar to human scrub typhus. Therefore, the use of O. tsutsugamushi Gilliam strain should be evaluated further in the rhesus macaque scrub typhus model in immunopathophysiological time-course studies of severe scrub typhus disease to support future vaccine development.

Abstract #49 Q fever Seroprevalence in Farmers, Wildlife Personnel and Ruminants in Thailand 2012 Kelly A. Fitzpatrick1*, Pawinee Doung-ngern2, Pattarin Opaschaitat3, DechaPangjai4, Pawin Padungtod5, and Gilbert J. Kersh1 1Rickettsial Zoonoses Branch, CDC, Atlanta, GA 2Bureau of Epidemiology, Department of Disease Control, Bangkok, Thailand 3National Institute of Animal Health, Bangkok, Thailand 4National Institute of Health, Bangkok, Thailand 5 Division of Global Health Protection, Thailand Ministry of Public Health-U.S.CDC Collaboration, Bangkok, Thailand

In a 2010-2012 study of infective endocarditis, Coxiella burnetii, the causative agent of Q fever, was found to be the most common zoonotic bacterium associated with endocarditis in Khon Kaen, Thailand. With this increased awareness of Q fever, further study of exposure to Coxiella burnetii was desired. Increasing understanding of the impact of Q fever in Thailand began with training to improve the laboratory capacity for detection and diagnosis. Instruction was given to staff at Thai veterinary and clinical laboratories including serological and molecular techniques as well as animal and environmental sampling. For this study Thai lab staff tested, milk, vaginal swab and environmental samples by PCR, sera by ELISA and IFA. Human sera were collected from those occupationally involved in ruminant care. Ruminant sera were collected from Chiang Mai, Nakhon Ratchasima, and Nakhonsi thammarat provinces. Of 1,632 ruminants tested in the sero-survey, cattle exhibited the highest prevalence at 4.6% (45/988), goats 3.5% (18/56) and sheep 2.1% (1/48). Nakhon Si Thammarat in the South had no cattle with antibodies to C. burnetii and only 3 goats and sheep (.46%), while cattle in Chiang Mai province in the North had the highest sero-prevelance at 5.5%. Nakhon Ratchasima, in northeastern Thailand, had high seroprevalence in goats and sheep (5.9%), but cattle were low (1%). Human seroprevalence in Chiang Mai was 42.8% (68/159) and in Nakhon Ratchasima was 2.99% (15/502), for a combined seroprevalence of 12.6% (83/661). Training of laboratory staff has increased the capacity of Thailand to evaluate Coxiella burnetii burden. Significant differences in seroprevalence were observed between geographic and between ruminant species. Each province reflected a different potential source of human infection. Human data indicates that farmers in Chiang Mai province are at high risk for seroconversion. Dairy cattle as opposed to other ruminants in that area appear to be the primary source of the bacteria.

45 Abstract #50 Optimization of ACCM for axenic growth of multiple genotypes of Coxiella burnetii Rachael Priestley* and Gilbert Kersh Rickettsial Zoonosis Branch, Centers for Disease Control and Prevention, Atlanta, GA

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular bacteria that can be challenging to grow in vitro. Common techniques for growing C. burnetii include cell culture and embryonated yolk sacs. Unfortunately both of these techniques can be very time and labor intensive, and they require purification of the organism following growth. The recent development of Acidified Citrate Cysteine Media (ACCM-2), has allowed for axenic growth of C. burnetii. However, we have found that not all genotypes of C. burnetii grow equally well in ACCM-2, including ST8, a genotype currently circulating in the US that has been implicated in recent outbreaks of Q fever. We have attempted to extend the utility of ACCM-2 by adjusting the conditions used when growing Coxiella in ACCM-2. With a slightly higher pH and longer time for growth, we have been able to increase the yields of several common genotypes of C. burnetii that previously were not favorable to axenic growth, without manipulating the published formula of the media. Using these newly optimized growth conditions we were able to achieve yields of the ST8 isolate GP-WA1 that are comparable to the growth of the ST16 isolate Nine Mile Phase 1, the isolate that was used in the development of ACCM-2. Furthermore, we are currently testing multiple genotypes of C. burnetii to evaluate their growth rates using these optimized conditions, with the goal of developing conditions suitable for growth of all known C. burnetii genotypes in ACCM-2.

Abstract #51 Characterization of RpoS in biphasic development and host cell colonization of Coxiella burnetii Derek E. Moormeier*, Diane C. Cockrell, Paul A. Beare, Kelsi M. Sandoz, and Robert A. Heinzen Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT

The obligate intracellular pathogen Coxiella burnetii is the causative agent of the zoonotic disease Q-fever. During host cell colonization, C. burnetii requires translocation of effector proteins into the host cell cytosol by the Dot/Icm type 4B secretion system (T4BSS). In addition, C. burnetii undergoes biphasic development producing physiologically and morphologically distinct large cell variant (LCV) and small cell variant (SCV) forms. The LCVs are replicating forms produced during the exponential growth phase, while SCVs are non-replicating, stress-resistant forms produced during the stationary growth phase. A similar biphasic developmental program in Legionella pneumophila is mediated by RpoS and stringent response physiology. In addition, recent transcriptomic profiling of C. burnetii SCVs revealed an increase in rpoS transcript levels relative to LCVs. Hence, we hypothesized that RpoS plays a role in regulating biphasic development and host cell colonization in C. burnetii. Here, we constructed a C. burnetii rpoS deletion mutant and examined its function in biphasic development, viability, intracellular growth, and gene regulation. By electron microscopy, the rpoS mutant grown to stationary phase exhibited unique morphological differences when compared to wild-type cells. This correlated with decreased viability, as assessed by an infectious focus-forming unit assay, and diminished intracellular growth, as assessed by genome equivalent measurements. Immunoblot analyses demonstrated that the chromatin-associated SCV-specific protein ScvA was not expressed in the rpoS mutant, and that the T4BSS apparatus protein IcmD, was down regulated. RNA-sequencing and whole cell mass spectrometry of exponentially and stationary phase grown wild-type and ΔrpoS cells are being conducted to reveal constituents of the RpoS regulon. Collectively, these data suggest that RpoS has a function in regulating biphasic development and host cell colonization.

Abstract #52 Characterization of PKC signaling during Coxiella burnetii infection C. Joel Funk1 and Daniel E. Voth2 1Dept. of Biology, John Brown University, Siloam Spring, AR 2Dept. of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR

C. burnetii infection of a macrophage results in internalization of the bacterial cell into a phagosome that converts to a lysosome-like organelle termed a parasitophorous vacuole (PV). This conversion from a phagosome into a PV requires 46 interruption and re-programming of normal cell processes, including signaling events. Previous evidence indicates activity of cellular kinases and phosphatases is critical for this transition from a phagosome to the PV. The role of Protein Kinase C (PKC) isoforms in PV generation and throughout the infection cycle was investigated using phospho-specific antibodies that recognize six isoforms expressed in macrophages. Western blot analysis indicated three isoforms, alpha, beta, and eta, were activated during infection of THP-1 macrophage-like cells. Further studies using small molecule inhibitors confirmed PKC-alpha involvement during PV formation. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a PKC substrate phosphorylated during infection. The location of MARCKS within infected cells was investigated using antibody- based localization and GFP-tagged MARCKS expression. Our results indicate that MARCKS localizes to the PV membrane and surrounding cytoplasm, indicating a possible role for MARCKS during infection. To investigate a possible function, MARCKS expression was targeted using siRNA treatment of HeLa cells. Infected cells treated with MARCKS siRNA contained C. burnetii, but many PV were larger in size and contained few bacteria compared to infected cells treated with non-targeting siRNA. A peptide inhibitor of MARCKS had a similar effect on infected THP-1 cells, resulting in enlarged PV compared to control peptide treatment. These results indicate that PKC activation and the PKC substrate MARCKS play a role in infection by regulating PV size.

Abstract #53 Human factor H-binding proteins of Bartonella bacilliformis and potential role in serum resistance Benjamin Mason1, Richard T. Marconi2, Linda D. Hicks1 and Michael F. Minnick1 1Division of Biological Sciences, University of Montana, Missoula, MT 59812 2Department of Microbiology & Immunology, Virginia Commonwealth University Medical Center, Richmond, VA

Bartonella are widespread bacterial pathogens of vertebrates and have been found to infect every type of mammal surveyed, to date. Of 31 validated species in the Bartonella genus, eleven are agents of serious infectious diseases of humans, including Carrión’s disease, trench fever, cat-scratch disease, and bacillary angiomatosis of AIDS patients. Bartonellae are facultative intracellular parasites that employ hemotrophy (infection of erythrocytes). Thus, a key trait for their survival is the ability to replicate within the bloodstream of a vertebrate host. Surprisingly, the molecular basis of serum resistance has not been previously determined for any Bartonella species. To that end, we determined that Bartonella bacilliformis, the agent of Carrión’s disease in humans and most virulent Bartonella species, is markedly resistant to human complement-mediated killing relative to E. coli. By using far-Western blots and total cell lysates, proteins of approximately 122, 89, 49, 32, and 20 kDa were identified as prominent human factor H-binding proteins (Fhbp’s). Immunofluorescence microscopy also showed strong binding of human factor H to the surface of intact B. bacilliformis as compared to bacterial cells treated with PBS or serum controls. Candidate genes for the 122-kDa and 89- kDa Fhbp’s were identified based upon the large, predicted molecular mass and putative surface location of their encoded polypeptides (BARBAKC583_0912 and BARBAKC 583_0512, respectively). Both genes encode putative trimeric autotransporter adhesins (TAAs) with homology to Vomp and Brp proteins of other Bartonella and YadA of Yersinia. These genes are being analyzed for activities related to human complement resistance.

Abstract #54 Robust growth of avirulent phase II Coxiella burnetii in bone marrow-derived murine macrophages Diane C. Cockrell1*, Jeffrey G. Shannon2, Shelly J. Robertson3, Heather E. Miller1, Lara Myers4, Carrie M. Long1, and Robert A. Heinzen1 1Laboratory of Bacteriology, Rocky Mountain Laboratories, Hamilton, MT; 2Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, Hamilton, MT; 3Laboratory of Virology, Rocky Mountain Laboratories, Hamilton, MT 4Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, Hamilton, MT

Published data show that primary mouse macrophages severely restrict growth of avirulent phase II, but not virulent phase I, Coxiella burnetii. This growth restriction results from potentiated recognition of pathogen-associated molecular patterns leading to generation of inflammatory TNF- medium (CM) from L929 murine fibroblasts as a source of macrophage-colony stimulating factor (M-CSF) to promote differentiation of bone marrow-derived myeloid precursors into macrophages. However, uncharacterized components of 47 CM and potential contaminants, such as lipopolysaccharide, can affect macrophage activation status and their permissiveness for infection. In the current study, we show that the Nine Mile phase II (NMII) strain grows robustly in primary macrophages from C57BL/6 mice when bone marrow cells are differentiated with recombinant murine M-CSF (rmM-CSF). Bacteria replicated within LAMP-1-postive vacuoles and achieved 2-3 logs of growth over 6 days. Similar results were obtained using C57BL/6 myeloid progenitors immortalized with an estrogen-regulated Hoxb8 (ER-Hoxb8) oncogene. To demonstrate the utility of the ER-Hoxb8 system, myeloid progenitors from natural resistance-associated macrophage protein (NRAMP)1 C57BL/6 knock-in mice were transduced with ER-Hoxb8, then macrophages were derived from the immortalized progenitors using rmM-CSF and infected with NMII. No difference in growth was observed when compared to macrophages from wild type mice, indicating depletion of metal ions by the NRAMP1 transporter does not negatively impact NMII growth. M-CSF differentiated murine macrophages from bone marrow and conditional ER-Hoxb8 myeloid progenitors will be useful ex vivo models for studying Coxiella-macrophage interactions.

Abstract #55 Anaplasma phagocytophilum AmpB is exposed on the cytosolic face of the pathogen-occupied vacuole and co-opts host cell SUMOylation Aminat T. Oki*, Bernice Huang, Andrea R. Beyer, Levi J. May, and Jason A. Carlyon Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA

Anaplasma phagocytophilum, a member of the family Anaplasmataceae and the obligate intracellular bacterium that causes granulocytic anaplasmosis, resides in a host cell-derived vacuole. Bacterial proteins that localize to the A. phagocytophilum-occupied vacuole membrane (AVM) are critical host-pathogen interfaces. Of the few bacterial AVM proteins that have been identified, their domains responsible for AVM localization and the host cell pathways that they co-opt are poorly understood. AmpB (A. phagocytophilum post-translationally modified protein B; formerly APH0032) is an effector that is expressed and localizes to the AVM late during the infection cycle. Herein, the AmpB domain that is essential for associating with host cell membranes was mapped to a 12-amino acid hydrophobic stretch. Immunofluorescent labeling of infected cells that had been differentially permeabilized confirmed that AmpB is exposed on the AVM’s cytosolic face, signifying its potential to interface with host cell processes. SUMOylation is the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates. Previous work from our laboratory determined that SUMOylation is important for A. phagocytophilum survival and that SUMOylated proteins decorate the AVM. Algorithmic prediction analyses identified AmpB as a candidate for SUMOylation. AmpB pronouncedly colocalized with SUMO-1 but not SUMO-2/3 moieties on the AVM. Endogenous AmpB was precipitated from infected cells using a SUMO affinity matrix, confirming that the effector is SUMOylated during infection. Ectopically expressed AmpB boosted the bacterial load, while a lysine-deficient version was less effective, implying that lysines contribute to AmpB function. This study delineates the first domain of any Anaplasmataceae protein that is essential for associating with the pathogen-occupied vacuole membrane, demonstrates the importance of AmpB to A. phagocytophilum infection, and identifies it as the second A. phagocytophilum effector that co-opts SUMOylation, thus underscoring the relevance of this post-translational modification to infection.

Abstract #56 Flotillins Associated with Cholesterol are Required and Recruited for Intracellular Proliferation of Anaplasma phagocytophilum Qingming Xiong and Yasuko Rikihisa* Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA

Anaplasma phagocytophilum is a cholesterol-dependent bacterium that proliferates in membrane-bound inclusions in granulocytes and causes human granulocytic anaplasmosis (HGA). Flotillin-1 and -2 are cholesterol-associated membrane proteins. In this study we found that A. phagocytophilum proliferation, but not entry is dependent on flotillin-1 and -2. Flotillin-1 and -2-containing vesicles were enriched and encircled A. phagocytophilum inclusions in host cells as early as 4 h post infection. This was validated on A. phagocytophilum inclusions in mCherry-flotillin-1 or GFP-flotillin-2-expressing cells. The flotillins containing vesicles surrounding A. phagocytophilum inclusions are loaded with free cholesterol indicated by cholesterol probes filipin and fluorescently-labeled theonellamides possessing aminomethyl coumarin acetic 48 acid (fTNM-AMCA) labeling. Localization of flotillins to A. phagocytophilum inclusions was dependent on cholesterol since this was abrogated by cholesterol-sequestering and lipid raft-disrupting agent M-β-cyclodextrin (MβCD) treatment and GFP-flotillin-2 bearing two mutations in cholesterol recognition/interaction amino acid motifs could not target the inclusions. Taken together, the data revealed that accumulations of flotillins on A. phagocytophilum inclusions is cholesterol-dependent and is crucial for A. phagocytophilum replication in the cells. Further, our results suggest ERK1/2 activation in A. phagocytophilum-infected cells is dependent on flotillins and cholesterol. Thus A. phagocytophilum mobilizes two critical signaling molecules: floillins and ERK1/2 via cholesterol for its benefit.

Abstract #57 Ehrlichia chaffeensis Nucleomodulin TRP32 is Regulated by Diverse Ubiquitin Modifications During Infection Tierra Farris1,2*, Bing Zhu2 and Jere W. McBride1,2 Departments of Pathology1 and Microbiology and Immunology2, University of Texas Medical Branch, Galveston, Texas Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes. Subversion of host post-translational modification (PTM) machinery including SUMO and ubiquitin to regulate ehrlichial effectors is one strategy used by E. chaffeensis. Previously we have shown that the E. chaffeensis TRP32 effector is a nucleomodulin which accesses the host nucleus via a tyrosine phosphorylation dependent mechanism and directly regulates transcription of specific host genes. In this study we investigated the role of ubiquitination in regulating TRP32 function and localization within the host cell. Ubiquitination is a complex and varied PTM that consists of either a single ubiquitin moiety, or one of several structurally and functionally distinct poly-ubiquitin chains and which controls several facets of protein function including trafficking, allosteric regulation, protein-protein interactions and proteasomal degradation. TRP32 contains four predicted ubiquitination sites, all within the TR DNA-binding domain; moreover, two sites are proximal to TRP32 putative transactivation domains. In order to determine the role of TRP32 ubiquitination on protein function, TRP32 ubiquitin modifications were characterized using immunoprecipitation and immunoblotting techniques. We found that TRP32 exists as a population of both mono- ubiquitinated and poly-ubiquitinated forms when ectopically expressed and during infection. Additionally, multiple mono- ubiquitinated TRP32 bands were observed during infection, which is consistent with TRP32 ubiquitination occurring at multiple resides. When the poly-ubiquitin chains were examined by immunoblotting, K63-linked poly-ubiquitin chains were detected, but it is likely that K48-linked chains are also present. Current work includes the use of TRP32 constructs in which specific lysine residues are mutated to identify the modifications that occur on specific residues and to determine the role of these modifications on TRP32 function and localization.

Abstract #58 The Tick Protein Sialostatin L2 Binds to Annexin A2 and Inhibits NLRC4-Mediated Inflammasome Activation Xiaowei Wang*, Dana K. Shaw and Joao H.F. Pedra Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21218, USA

Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that the Loop2 of sialostatin L2, an anti-inflammatory tick protein, binds to Annexin A2, and impairs formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum. Macrophages deficient in Annexin A2 secreted significantly lower amounts of interleukin (IL)-1β and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia and monocytopenia. Providing translational support to our findings, better binding of Annexin A2 to sialostatin L2 was also demonstrated in the sera from 21 out of 23 infected patients when compared to control individuals. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.

49 Abstract #59 “MicroRNA signature” of human microvascular endothelium infected in vitro with Rickettsia rickettsii Abha Sahni*, Hema P. Narra, Jignesh Patel, and Sanjeev K. Sahni Department of Pathology and Institute for Human Infections and Immunity, The University of Texas Medical Branch

Infection of human microvascular endothelial cells (HMECs) as preferred, primary targets of pathogenic Rickettsia species triggers host responses via activation of immediate-early signaling cascades and subsequent induction of gene expression. MicroRNAs (miRNAs) are important regulators of gene expression in the immune system and miRNA-mediated RNA interference is now regarded as a critical regulatory mechanism operating at the level of translation. Herein, we report our findings of miRNA profiling of HMECs infected with Rickettsia rickettsii using human Affymetrix GeneChip® miRNA microarray. For identification of differentially expressed miRNAs, total RNA from R. rickettsii-infected and corresponding mock-infected HMECs was isolated using TRI-reagent®, following which array hybridization::scanning procedures were performed according to the Affymetrix miRNA protocol. Analysis of microarray data revealed miRNAs-129-5p, 297, 595, 574-5p, 647 as the top five up-regulated miRNAs (5 to 20-fold, p≤ 0.01) at 3 and 24h post-infection. Also, several other miRNAs, including miRNA 146a, 631, 661, and 766, were up-regulated only at 24h post-infection (4 to 13-fold, p≤0.01). Conversely, miRNAs 301b, 548a-3p, and 377 were significantly down-regulated (2 to 3-fold, p≤ 0.05) at 3 and 24h, whereas miRNAs 376b, 216a, 216b, 410, and 29b were all down-regulated at 24h post-infection. The microarray expression data was further consolidated via confirmation of four significantly up-regulated miRNAs by quantitative PCR, which suggested even higher degree of induction for miRNA 129-5p, 297, 595, and 574-5p (≥20-fold) in R. rickettsii-infected HMECs. Using the microRNA database miRBase, we have further identified both validated as well as predicted gene targets for miRNAs displaying robust changes during R. rickettsii infection of HMECs. From this preliminary analysis arises the first evidence for a significantly altered ‘miRNA expression pattern’ during rickettsial infection of host cells. Ongoing studies on miRNA- governed regulation of gene expression are expected to provide new perspectives on the host response and pathogenesis mechanisms during human rickettsioses.

Abstract #60 Mechanisms of Non-canonical Inflammasome Activation by Rickettsia C. Smalley*, J. Bechelli, D.H. Walker, R. Fang 1 University of Texas, Medical Branch, Department of Pathology

Rickettsiae are Gram-negative, obligately intracellular bacteria that infect macrophages. However, the molecular mechanisms involved in interaction of rickettsiae with macrophages remain poorly understood. In this study, we investigated the non-canonical inflammasome activation by rickettsiae in mouse macrophages. R. australis activated the non-canonical inflammasome in mouse bone-marrow derived macrophages (BMDM), marked by caspase (casp)-11 activation and the induction of pyroptosis. Here, we have investigated the role of rickettsial lipopolysaccharide (LPS) in activating the caspase-11 dependent non-canonical inflammasome. Taken together, our data, for the first time, illustrates the role of caspase-11 in activation of inflammasome by rickettsiae in mouse macrophages. This study provides invaluable information for developing novel therapeutic interventions for rickettsioses by employing current knowledge derived from mouse infection models.

Abstract #61 The Rickettsia conorii Adr2 promotes resistance to complement-mediated killing Daniel A. Garza*, Sean P. Riley, and Juan J. Martinez Vector-Borne Diseases Laboratories, Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA, 70803, USA

All bacteria exposed to host serum are subject to the antibacterial effects to the complement system. However, many bacteria have evolved mechanisms of evading this immune attack. We have previously demonstrated that R. conorii Adr1 and OmpB β-peptide contribute to complement evasion by binding the complement regulatory proteins vitronectin and factor H. Since Adr2 shares a conserved structure with Adr1 and both proteins were demonstrated to interact with an unknown host surface proteins, we hypothesized that Adr2 may contribute to the rickettsial complement evasion 50 phenotype. In this study, we utilize a heterologous system to express Adr2 at the surface of serum-sensitive E. coli to examine the potential role of this protein in the evasion of complement-mediated killing and adherence to host cells. Through expression of Adr2 in E. coli, we demonstrate that this protein is capable of conferring both serum resistance and the ability to interact with the host complement regulator vitronectin. Additionally, we demonstrate that expression of various rickettsial Adr2 proteins is insufficient to mediate adherence to cultured mammalian endothelial cells. These data further our understanding of rickettsial complement evasion phenotypes and contradict previous hypotheses that Adr2 contributes to interaction with the host cell.

Abstract #62 Involvement of Pore Formation and Osmotic Lysis in the Rapid Killing of Gamma Interferon-Pretreated, C166 Endothelial Cells by Rickettsia prowazekii Jenifer Turco Department of Biology, Valdosta State University, Valdosta, GA 31698

The cause of epidemic typhus is Rickettsia prowazekii, an obligate, intracellular bacterium that grows primarily within the microvascular endothelial cells. Endothelial cell damage is a prominent feature of R. prowazekii infections in vivo. Earlier studies with cultured, murine macrophage-like RAW264.7 cells revealed that the combination of pretreating the macrophages with gamma interferon (IFN-) and then infecting them with R. prowazekii causes many of the macrophages to die (become permeable to trypan blue) within several hours after infection. The present study was undertaken to determine if cultured, murine C166 endothelial cells would be similarly damaged and to evaluate the possible roles of pore formation and osmotic lysis in host cell death. Within several hours after the addition of R. prowazekii, many of the IFN--pretreated C166 endothelial cells became permeable to trypan blue (961 daltons) and ethidium bromide (394 daltons); in addition, substantial amounts of lactate dehydrogenase (LDH, approximately 140 kilodaltons) were released into the culture media. In contrast, similar evidence of cell death was not observed in untreated, infected endothelial cell cultures, or in mock-infected cultures, whether IFN--pretreated or untreated. To determine if pore formation and osmotic lysis contributed to endothelial cell death, the ability of polyethylene glycols (PEGs) of various molecular weights to protect the cells was assessed. Other researchers have used PEGs to estimate the size of membrane pores in damaged cells. PEGs are osmoprotectants that can prevent osmotic cell lysis and the associated release of LDH if they are large enough to be retained outside of the initially damaged cells. In the present study, PEGs with nominal average molecular weights of 8,000 (PEG 8000, 15 mM) dramatically suppressed the release of LDH; in contrast, PEG 4000 (30 mM) only partially inhibited LDH release, and PEGs 2000 and 1450 (30 mM) had little or no effect on LDH release. Although PEG 8000 inhibited LDH release from the IFN--pretreated, infected C166 cells, it did not prevent their uptake of ethidium bromide. These findings suggest that pore formation and osmotic lysis contributed to the death of the IFN--pretreated, R. prowazekii-infected endothelial cells. Based on the hydrodynamic radii of PEG 2000 (1.4 nm) and PEG 8000 (3.2 nm), one can estimate that the pore diameters in the damaged, IFN--pretreated, infected endothelial cells were larger than 2.8 nm and smaller than 6.4 nm. The results of this study are consistent with the hypothesis that IFN-, which is an important anti-rickettsial host defense, may also enhance the ability of R. prowazekii to damage endothelial cells.

Abstract #63 Autophagy Promotes Rickettsia australis Replication in Primary Murine Macrophages Jeremy Bechelli1*, Claire Smalley1, Xuemei Zhao1, Seungmin Hwang2, David H. Walker1, Rong Fang1 1Department of Pathology, University of Texas Medical Branch at Galveston. 2Department of Pathology, University of Chicago

Rickettsiae are obligately intracellular bacteria that replicate in the cytoplasm of eukaryotic cells. We found autophagy induction and co-localization of bacteria with LC3 (+) autophagosomes in murine bone marrow-derived macrophages (BMMs) upon infection with R. australis. Autophagy induction by rickettsiae in BMMs of Atg5flox/flox mice was completely abrogated in the counterparts of Atg5flox/flox Lyz-Cre animals. Rickettsial replication was significantly greater in BMMs of Atg5flox/flox mice compared to Atg5flox/flox Lyz-Cre mice, suggesting that Atg5-dependent autophagy promotes rickettsial growth in primary mouse macrophages. Autophagy inducers significantly promoted rickettsial replication in primary macrophages compared to untreated controls. Furthermore, R. australis-infected Atg5flox/flox macrophages secreted 51 significantly lower levels of pro-inflammatory cytokines including inflammasome-dependent IL-1β compared to Atg5flox/flox Lyz-Cre macrophages. Strikingly, rickettsial growth in Atg5flox/flox macrophages was unchanged from that in Atg5flox/flox Lyz- Cre macrophages when cells were treated with neutralizing antibodies against IL-1β, suggesting that IL-1β was involved in greater bacterial replication in autophagy-competent cells. In line with the in vitro results, the in vivo rickettsial loads in liver, lung and spleen of infected Atg5flox/flox mice were significantly higher than those in Atg5flox/flox Lyz-Cre mice accompanied by a significantly lower level of IL-β in the serum. Taken together, our data demonstrated the role of autophagy in promoting rickettsial replication in mouse macrophages both in vitro and in vivo.

Abstract #64 Post-translational modification of the immunodominant autotransporter, OmpA in Rickettsia rickettsii Nicholas F. Noriea*, Tina R. Clark, and Ted Hackstadt Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountains Labs, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, contains two immunodominant proteins, rOmpA and rOmpB, in the outer membrane. These proteins make up a significant portion of the outer membrane proteome and S-layer. Both rOmpA and rOmpB are conserved throughout the spotted fever group rickettsia as members of a family of autotransporter proteins called surface cell antigens (sca). Previously, it was demonstrated that rOmpB is proteolytically processed with the cleavage site residing near to the autotransporter domain at the carboxy-terminal end of the protein, cleaving the 168 kDa protein into apparent-sized 120 kDa and 32 kDa fragments. The 120 and 32 kDa fragments remain non-covalently associated on the surface of the bacterium with implications that the 32kDa fragment functions as the membrane anchor domain. Here we present evidence for a similar post-translational processing of rOmpA. Surface proteome analysis via mass spectrometry has identified a small fragment rOmpA corresponding to the carboxy terminal end of the protein downstream of the predicted cleavage site. A rabbit polyclonal antibody specific to the autotransporter region of rOmpA identified a peptide with a mass of ~30 kDa corresponding to the calculated mass of a proteolytically cleaved rOmpA autotransporter region. Available mass spectrometry data and protein analysis has demonstrated that the putative rOmpA c-terminal fragment is present in all virulent strains of R. rickettsii to date, but absent from the avirulent strain ‘Iowa’.

Abstract #65 Characteristics of unique Rickettsia rickettsii Iowa clones Tina R. Clark*, Nick Noriea, Ted Hackstadt 1Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana

Rickettsia rickettsii Iowa is an avirulent spotted fever group rickettsia that has displayed marked changes in virulence since its original isolation. It has varied from mildly virulent to virulent to avirulent over many egg passages. One explanation for the variation in virulence of R. rickettsii Iowa is that the original seeds were mixed cultures containing both virulent and avirulent clones. To test this, an early seed from egg passage four was plaqued and examined for differences. Two distinct plaque morphologies were observed; a large opaque plaque (L) and a small clear plaque (S). Both were cloned and expanded for further evaluation. The L clone displayed a more rapid growth rate than the S clone and exhibited the same characteristics as the high passage R. rickettsii Iowa described previously. These included a processing defect in rOmpB and the absence of rOmpA. The S clone also displayed many of the same characteristics as R. rickettsii Iowa, but unlike L clone expresses rOmpA. To confirm that both the L and S clones were R. rickettsii Iowa, multiple SNPs unique to the Iowa strain were sequenced and compared to R. rickettsii Iowa. The similarity of these sequences suggests that both clones are of the R. rickettsii Iowa lineage. The isolation of clonal variants of R. rickettsii Iowa that differ in the presence of rOmpA has permitted the evaluation of the role of rOmpA in virulence and processing of the autotransporter. Neither variant was virulent in a Guinea pig model of infection. The rOmpA precursor, like rOmpB, was not efficiently processed by the Iowa small plaque variant.

52 Abstract #66 Ehrlichia chaffeensis effector driven reprogramming of the macrophage Jere W. McBride Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

Ehrlichia chaffeensis is an obligately intracellular bacterium that has evolved molecular strategies to hijack the mononuclear phagocyte and avoid destruction by innate defense mechanisms. Recently, our laboratory has identified a group of type 1 secreted tandem repeat protein (TRP) effectors, and uncovered an array of diverse molecular interactions between these effectors and specific host cell targets that are important for ehrlichial survival. Host cell processes and signaling pathways targeted by E. chaffeensis TRP effector interactions are essential for normal cell function and are highly evolutionarily conserved. Studies to define the functional significance of these pathogen-host interactions has revealed that E. chaffeensis TRPs moonlight and have different functional roles during infection depending on their cellular location. TRP effectors exploit host post translational modification (PTM) pathways to obtain modifications such as phosphorylation, SUMOylation/ubiquitination, which modulate effector function, subcellular trafficking, and influence effector-host interactions, thereby modulating cellular processes to promote survival. The moonlighting effector, TRP120, is involved in host cell entry, has Ub ligase activity, and is also a nucleomodulin that translocates to the host cell nucleus where it reprograms host cell gene transcription through direct and epigenetic mechanisms. Host cell pathways that E. chaffeensis exploits to promote survival include, Wnt, Notch, and Sonic Hedgehog in an overall strategy to prolong host cell survival and to avoid innate immune defenses.

Abstract #67 Rickettsia parkeri harnesses intercellular tension forces to promote cell-to-cell spread Rebecca L. Lamason1, Effie Bastounis2, Natasha M. Kafai1, Ricardo Serrano3, Juan C. del Álamo 3, 4, Julie A. Theriot2, Matthew D. Welch1 1 Department of Molecular and Cell Biology, University of California, Berkeley; 2 Departments of Biochemistry, Microbiology and Immunology and Howard Hughes Medical Institute, Stanford University School of Medicine; 3 Mechanical and Aerospace Engineering Department, University of California, San Diego; 4 Institute for Engineering in Medicine, University of California, San Diego

Spotted fever group (SFG) rickettsiae disseminate through host tissues by a process of cell-to-cell spread that involves protrusion formation, engulfment, and vacuolar escape. Other bacterial pathogens rely on actin-based motility to provide a physical force for spread. Although SFG rickettsiae undergo actin-based motility driven by either RickA or Sca2, we show that the SFG species Rickettsia parkeri does not appear to harness actin-driven pushing forces during spread. Instead, they manipulate host intercellular tension and mechanotransduction to promote cell-to-cell transfer. Using transposon mutagenesis, we identified surface cell antigen 4 (Sca4) as a secreted effector of spread that specifically acts in the donor cell to promote protrusion engulfment. Sca4 interacts with the cell adhesion protein vinculin and blocks association with -catenin. Using single cell traction force microscopy and monolayer stress microscopy, we show Sca4 reduces vinculin-dependent mechanotransduction at cell-cell junctions. Our results suggest that Sca4 relieves intercellular tension to promote protrusion engulfment, which represents a distinctive strategy for manipulating cytoskeletal force generation to enable spread.

Abstract #68 Anaplasma phagocytophilum parasitizes host acid sphingomyelinase for completion of its infection cycle Chelsea L. Cockburn* and Jason A. Carlyon Department of Microbiology & Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA.

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils and causes the potentially fatal febrile illness, human granulocytic anaplasmosis (HGA). It undergoes a biphasic developmental cycle, transitioning between non-infectious, replicative reticulate cell (RC) and non-replicative, infectious dense core (DC) forms. We recently reported that A. phagocytophilum intercepts exocytic traffic from the trans-Golgi to acquire sphingolipids and convert to 53 the DC form. Sphingolipidomic analyses of DCs revealed high amounts of C16-ceramide. We examined the hypothesis that C16-ceramide is crucial for A. phagocytophilum development. Acid sphingomyelinase (ASMase), a host cell enzyme that hydrolyzes sphingomyelin into C16-ceramide, localized to the A. phagocytophilum vacuole membrane and associated with the bacteria by 12 h post-infection. In infected host cells, smpd1, the gene that codes for ASMase, was significantly upregulated when compared against uninfected control cells. Inhibition of ASMase enzymatic activity resulted in arrest of the A. phagocytophilum infection cycle after 24 h. Additionally, A. phagocytophilum RC-to-DC conversion was significantly reduced in ASMase inhibited cells. Overall, these data indicate that ASMase is critical for progression of the A. phagocytophilum infection cycle, specifically for RC-to-DC conversion. This is the first example of host ASMase and C16- ceramide being necessary for the infection cycle of any obligate intracellular bacterium and provides insight into how and why these organisms parasitize host sphingolipids.

Abstract #69 Metabolic and biosynthetic activity of Ehrlichia chaffeensis in a host cell-free medium Vijay K. Eedunuri1, Chuanmin Cheng1, Anders Omsland2, Dan Boyle3, and Roman R. Ganta1* 1Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506; 3Paul G. Allen School for Global Animal Health, PO Box 647090, Washington State University, Pullman WA 99164; 2Division of Biology, Kansas State University

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis (HME). Biochemical characterization of this pathogen remained a major challenge, as the organism requires a host cell for its replication. In particular, culturing E. chaffeensis and the related rickettsials outside a host cell are not yet described. Recently, Omsland et al. described an axenic (host cell-free) nutrient medium that supports metabolic activity of Chlamydia trachomatis, another intracellular bacterium that undergoes a biphasic developmental cycle with infectious form referred as the elementary body (EB) and an intracellular replicative body (RB). The axenic culture system supports the metabolic and biosynthetic activity of both EBs and RBs with different nutrient requirements when incubated under microaerobic conditions. In the current study, we tested the hypothesis that E. chaffeensis having similar biphasic developmental cycle with infectious form referred as dense core cells (DCs) and the replicating form known as reticulate cells (RCs) can be cultured in a host cell-free medium under microaerobic conditions. E. chaffeensis harvested from the canine macrophage cell line (DH82) were fractionated into DCs and RCs by renografin density gradient centrifugation and propagated in the axenic culture medium containing a cocktail of intracellular phosphate buffer with the 20 amino acids and four nucleotides. Various carbon sources were also included. Incorporation of 35S Cysteine and Methionine into the bacterial proteins was observed primarily in the RCs of E. chaffeensis when glucose-6-phosphate was added to the media. ATP, alpha ketoglutarate or sodium acetate also supported the protein synthesis, but to a lesser degree. The metabolic and biosynthetic activity could not be detected in the DCs when cultured in the same axenic media for E. chaffeensis. The axenic media-specific protein synthesis in RCs was confirmed by including chloramphenicol as a protein synthesis inhibitor. The recovery of RCs and DCs of E. chaffeensis were verified by performing various experiments, including defining their inability/ability to infect host cells, their morphology by transmission electron microscopy and by performing Western blot analysis. The application of the axenic culture system will be valuable in furthering our understanding of the Ehrlichia metabolism and in defining the essential nutrient requirements. The methods described in our studies are also valuable in performing similar studies in the related Anaplasmataceae pathogens. (This work was supported by PHS grant number AI070908 from the National Institute of Allergy and Infectious Diseases, USA. This work also received support from the CEVBD, Kansas State University.)

Abstract #70 E. chaffeensis induced Wnt-mTOR signaling regulates transcription factor TFEB to inhibit autophagy. Taslima T. Lina, Tian Luo, Jere W. McBride* Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA.

Ehrlichia chaffeensis manipulates host cellular processes to create a favorable environment by reprogramming cell- signaling pathways and inhibiting bactericidal activity, most likely through specific interactions of its surface-expressed and/or secreted effector proteins. In multicellular organisms, autophagy is normally induced during a bacterial infection. 54 However, E. chaffeensis resides in late endosome that fail to fuse with lysosomes through an unknown mechanism. We detected the presence of autophagosome markers such as LC3 and Beclin-1 on the ehrlichial vacuoles. In contrast to the diffused distribution of LC3 (cytosolic LC3) in THP-1 cells, LC3 formed distinct punctate dots (membrane bound LC3II) in infected cells. However, co-localization of the ehrlichial vacuole with the lysosomal marker LAMP-2 was not observed. Using pharmacological inhibitor (Dvl-PDZ domain inhibitor, 3289-8625) we demonstrated that Wnt signaling plays a crucial role in inhibition of phagolysosomal fusion and lysosomal degradation. Underlying mechanisms involves Wnt mediated inhibition of glycogen synthase kinase-3 (GSK3) and activation of mTOR pathway. We demonstrated, activation of mTOR signaling by E. chaffeensis inhibits nuclear localization of transcription factor EB (TFEB), which is the master regulator of lysosomal biogenesis and phagolysosomal fusion. In addition, induction of GSK3 and inhibition of mTOR using small molecular inhibitor (Pyrivinum) significantly decreased ehrlichial load. Our data suggests that, activation of Wnt pathway contributes to the ability of E. chaffeensis to inhibit autophagy by reprogramming this host signal transduction pathway to enhance intracellular survival. This study reveals ehrlichial exploitation of a novel regulatory network, linking conserved Wnt pathway with autophagy.

Abstract #71 Targeted mutagenesis in Ehrlichia chaffeensis by homologous recombination Ying Wang*, Chuanmin Cheng and Roman R. Ganta Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66503

Targeted mutagenesis of Ehrlichia species and in related rickettsiale pathogens remains a big challenge. In a recent study, we reported that it is possible to create mutations in E. chaffeensis by employing both random and targeted mutagenesis. Stable mutations are documented at several genomic locations with the Himar I transposase-mediated random mutagenesis approach and that they are valuable in defining the requirement of a gene in a host. Targeted mutations are also created at several locations and they survived only for a few days under in vitro culture conditions. Further, our previous studies revealed that over 94% of the predicted genes of the organism are transcribed when cultivated in vitro in vertebrate macrophages and tick cells. Based on these data, we hypothesized that it is a challenge to mutate a specific gene of interest in E. chaffeensis, as its genome has fewer redundant spots where targeted gene disruptions are possible. Further, we postulated that targeted mutagenesis is likely to be accomplished if we select a genomic region identified as mutatable by random mutagenesis. To test these hypotheses, we chose two genes to create targeted mutations, Ech_0230 and Ech_0379, where we previously demonstrated insertion mutations by random mutagenesis. Plasmid constructs were prepared to include two fragments of DNA spanning the genes as 5’ and 3’ flanking sequences with a middle insertion containing the aadA gene to confer resistance to spectinomycin/streptomycin antibiotics. We engineered the E. chaffeensis tuf gene promoter to drive the expression of the aadA gene expression. The entire segments were then amplified from the plasmids as linear fragments and used to electroporate into cell-free E. chaffeensis organisms. The transformed bacteria were propagated in ISE6 tick cells in the presence of antibiotics to assess the homologous recombination. After two weeks, the antibiotic resistant organisms were observed. The presence of insertions in the E. chaffeensis genome was confirmed by PCR and sequence analysis targeting to the sequences flanking both the 5’ and 3’ to the insertion sites, and by Southern blot analysis using genomic DNA from the mutants. The targeted insertion mutants remained in culture stably for over two months and also grew well in the canine macrophage cell line, DH82. This is the first evidence demonstrating the generation of stable mutations by homologous recombination in E. chaffeensis. Targeted mutagenesis will aid in the efforts to create additional mutations in E. chaffeensis and in other related Anaplasmataceae pathogens. It will also be a valuable tool in devising inducible gene disruptions and in studying the specific gene function, pathogenesis and in developing effective methods of controlling the rickettsial infections. (This work was supported by the PHS grant # AI070908 from the National Institute of Allergy and Infectious Diseases, USA. This work also received support from the CEVBD, Kansas State University.)

55 Abstract #72 Monovalent Cation/Sodium:Proton Antiporter Proteins of Ehrlichia chaffeensis Lanjing Wei*, Huitao Liu, and Roman R. Ganta Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66503

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis (HME). Little is known about how this organism is able to reside and replicate within an acidified phagosome (~pH 5). Similarly, it is unclear how the infectious form of the bacterium maintains homeostasis in the extracellular milieu where the pH is ~7.35-7.45. Our central hypothesis is that E. chaffeensis regulates its intracellular pH with the help of its membrane bound monovalent cation:proton antiporter proteins. Sodium/cation:proton antiporters are integral membrane proteins that exchange sodium or other monovalent cations against protons across a plasma membrane in maintaining the cytoplasmic pH of a cell. We recently described a mutation within the Ech_0379 gene of E. chaffeensis predicted to encode for a Na+/H+ antiporter protein. The mutation caused attenuated growth of the organism in vertebrate hosts, but it persisted at lower level compared to wild type in vertebrate hosts (deer and dogs). Based on these data, we hypothesized that E. chaffeensis uses two or more antiporter proteins to regulate its intracellular pH. Indeed, the E. chaffeensis genome contains coding sequences encoding for 10 antiporter proteins or its subunits; based on their genomic location, we predicted them as encoding for 6 functional proteins. To define their function, we performed experiments in the Escherichia coli mutant strain (EP432); it has mutations at two of the three antiporter protein genes, thus making it sensitive to sodium. The growth of this strain can be restored if complemented with a heterologous antiporter gene. E. chaffeensis genes encoding for the predicted antiporter proteins were cloned with their respective native promoters into the plasmid, pBlueScript II SK+, and it was transformed into EP432. The transformants were then assessed to determine if they restore the growth of the E. coli under different Na+ concentrations. Two genes restored the growth similar to a positive control where the E. coli Na+/H+ antiporter (NhaA) is expressed. On the contrary, complementation with four predicted gene units made the E. coli to be more sensitive to Na+. These data suggest that E. chaffeensis antiporter proteins may function in regulating its intracellular pH. Studies are now underway in defining the role of E. chaffeensis antiporters. (This work was supported by PHS grant number AI070908 from the National Institute of Allergy and Infectious Diseases, USA.)

56

th 28 Meeting of the

American Society for Rickettsiology

Monday June 13, 2016

Abstracts #73-138

Vector Biology & Vector-Pathogen Interactions

Infection, Diagnosis, and Treatment

Poster Session 2

Funding for this conference was made possible [in part] by R13 AI126727-01 from the National Institute of Allergy and Infectious Diseases. The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the U.S. Department of Health and Human Services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government.

57 Abstract #73 Infection-Derived Lipids Elicit a Novel Immune Deficiency Circuitry in Arthropods Joao Pedra Department of Microbiology and Immunology, University of Maryland School of Medicine

Arthropod-borne illnesses account for approximately 17% of all infectious diseases. The urgency for understanding and combating vector-borne disease transmission is illustrated by the recent outbreaks of Dengue and Zika viruses in Latin America. Central to microbial dissemination in the arthropod vector is the Immune Deficiency (IMD) pathway, which senses Gram-negative bacteria and mounts a humoral response upon infection. The IMD signaling cascade is functionally analogous to the mammalian tumor necrosis factor (TNF) receptor network. In the classical mode, Gram-negative bacteria are recognized by the receptors PGRP-LC and PGRP-LE, which relay the immune signal through a series of post- translational modifications leading to production of anti-microbial peptides. In this lecture, I will talk about a novel IMD pathway molecular circuit in the tick Ixodes scapularis that protects against colonization by two evolutionarily distinct bacteria: the Lyme disease agent Borrelia burgdorferi and the rickettsial pathogen Anaplasma phagocytophilum. This biochemical circuit is elicited by infection-derived lipids and ensues independently of the death domain protein FADD and the adaptor molecule IMD. Conceptually, we will propose the existence of two functionally distinct IMD networks: one termed classical and occurring mostly in insects, and another exerted through lipid sensing displayed in ticks.

Abstract #74 One of the good catch! Fishing vector to study tick-pathogen interactions Girish Neelakanta Center for Molecular Medicine, Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529

In the United States, Ixodes scapularis ticks transmit various pathogens to humans that include Anaplasma phagocytophilum, the agent of Human anaplasmosis. Ticks ingest A. phagocytophilum while feeding on an infected animal. Upon entering, A. phagocytophilum establishes itself in the salivary glands and then transstadially maintained through different developmental stages of these ticks. Survival in the arthropod host throughout the different development stages is particularly important for a tick-borne pathogen such as A. phagocytophilum, which cannot be transovarially transmitted. Our previous studies have elucidated that A. phagocytophilum manipulates arthropod cell signaling to surive in its vector. In the view for the development of an anti- vector vaccine as an effective means to block transmission of A. phagocytophilum from these ticks, understanding the role of conserved arthropod molecules in vector biology and interactions with pathogens remains important. Organic anion transporters (OATPs) are one of the highly conserved protein families across various arthropod species. This study provides evidence for the role of OATPs in tick-A. phagocytophilum interactions. RNAi knockdown studies and in vitro cell culture experiments elucidate role of OATPs in A. phagocytophilum transmission and surival in these ticks. Collectively, this study defines a new pathway not only to understand vector biology but also may lead for the development of better stratagies to block transmission of A. phagocytophilum and perhaps other Rickettsial species from these ticks.

Abstract #75 Bartonella quintana utilizes a protein partner switch mechanism to survive stressful arthropod conditions Henriette Macmillan1, Sally Lyons-Abbott2,3, David M. Dranow3,4, Gina M. Borgo1, James W. Fairman3,4, Stephanie J. Huezo1, Donald D. Lorimer3,4, Bart L. Staker2,3, Robin Stacy2,3, Stephanie Abromaitis1,5, Thomas E. Edwards3,4, Peter J. Myler2,3,6, Jane E. Koehler*1 1Microbial Pathogenesis and Host Defense Program, and Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143, USA; 2Center for Infectious Disease Research, formerly Seattle Biomedical Research Institute, Seattle, WA 98109, USA; 3Seattle Structural Genomics Center for Infectious Disease, Seattle, WA, 98109; 4Beryllium, Bainbridge Island, WA 98110, USA; 5Current address: California Department of Public Health, Richmond, CA 94804, USA; 6Departments of Global Health and Biomedical Informatics and Health Education, University of Washington, Seattle, WA, 98195

58 The general stress response (GSR) is a widely conserved response utilized by bacteria to survive under a variety of extreme environmental conditions. The alpha-‐‐proteobacterium, Bartonella quintana, occupies two distinct niches: the bloodstream of the human host (37°C, severely restricted hemin levels) and the gut of the human body louse vector (28°C, highly toxic hemin levels). We previously identified an extracytoplasmic function (ECF) sigma (σ) factor, RpoE, which is involved in the GSR of B. quintana. Once in the louse midgut, B. quintana uniquely activates the GSR in response to the decreased temperature (28°C). During this transition from the human host to the body louse vector, a sensor histidine kinase phosphorylates an anti-‐‐anti-‐‐σ factor, PhyR. Phosphorylated PhyR then competes for, and removes, the anti-‐‐σ factor NepR from the RpoE σ factor. RpoE is then released and able to bind RNA polymerase core enzyme, thus enabling transcription of the regulon necessary for survival under body louse conditions. To identify the structural basis of B. quintana GSR regulation in the body louse, we solved the crystal structures of the RpoE-•‐NepR complex, the NepR-•‐PhyR complex, and unbound, unphosphorylated PhyR. These crystal structures revealed a dramatic conformational change in PhyR after phosphorylation, which enables PhyR binding of NepR using a partner-•‐switch mechanism. We also identified a putative sensor histidine kinase (BQ-•‐SHK) that phosphorylates PhyR. Additionally, the B. quintana BQ-•‐SHK null mutant was unable to activate the GSR in response to the louse low temperature stress signal of 28°C in vitro. The importance of RpoE was confirmed in vivo by demonstrating that a B. quintana rpoE null mutant is unable to multiply in the body louse. Our findings suggest that RpoE, BQ-•‐ SHK, and the conformational change of PhyR after phosphorylation, all have critical roles in the adaptive response of B. quintana to low temperature stress in the body louse arthropod vector.

Abstract #76 De novo folate biosynthesis of Rickettsia Endosymbiont Ixodes pacificus, a new Rickettsia species isolated from Ixodes pacificus Jianmin Zhong*, Maryam Alowaysi, Jimmy Bodnar, Kristine Teague, and Junyan Chen Department of Biological Sciences, Humboldt State University, Arcata, CA 95521

Nonpathogenic bacterial endosymbionts have been shown to contribute to their arthropod hosts’ fitness by supplying them with essential vitamins and amino acids. Little is known about the nutritional basis for the symbiotic relationship of endosymbionts in ticks. Recent metabolic reconstructions in our laboratory have shown that Rickettsia Endosymbiont Ixodes pacificus (REIP) carries all genes for de novo folate (vitamin B9) synthesis. In this study a culture isolate of REIP, obtained from ovaries of engorged I. pacificus, was used. Transmission electron microscopy showed that the bacterial morphology resembles typical Rickettsia species. Sequencing alignments, BLAST, and phylogenetic analysis of the six rickettsial genes (16S rRNA, gltA, ompA, dksA-xerC, mppA-purC and rpmE-tRNAfMet intergenic spacers) demonstrated that the isolate fulfills the criteria to be classified as a novel Rickettsia species that is closely related to Rickettsia monacensis and Rickettsia buchneri. In addition, we amplified all five rickettsial folate genes (folA, folC, folE, folKP, and ptpS) of REIP by PCR, cloned into an expression vector, and transformed into expression host BL21(DE3). Sequencing and bioinformatic analyses have identified the putative promoter and ribosome binding site as well as the functional domain of the open reading frame for each folate gene. SDS-PAGE results showed that all five recombinant folate proteins were overexpressed in BL21(DE3). Folate proteins were purified by either affinity column chromatography or batch method. In vitro enzyme assays using purified proteins detected that FolA and FolE protein of REIP has dihydrofolate reductase (specific activity of 16.1 U/mg) and GTP cyclohydrolase I activity (specific activity of 7.9 U/mg), respectively, both of them are required for folate biosynthesis. Furthermore, RT-qPCR assay detected that the folate genes (folA, folE, and folKP) in the folate biosynthesis pathway are expressed in REIP-infected ISE6 cells. However, the folate genes are not differentially expressed (P≥0.05) between 25°C and 36°C in REIP-infected ISE6 cells. Overall, our data suggest the nutritional interactions of the symbiosis between REIP and I. pacificus.

Abstract #77 Molecular Mechanism of Wolbachia Induced Cytoplasmic Incompatibility John F. Beckmann*, Judith A. Ronau, Mark Hochstrasser Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511

59 Cytoplasmic Incompatibility (CI) is an insect sterility induced by the obligate intracellular bacterium Wolbachia. Wolbachia appear to secrete a toxin into sperm causing deadly missegregation of paternal chromosomes in embryos. Similar to a toxin-antidote system, if a modified spermatid fertilizes an egg hosting the same Wolbachia symbiont, an antidote can rescue the incompatibility. Insects vector diseases as well as cause economic damage in agriculture and thus developing new tools to control their populations is an important task. Here we demonstrate that a Wolbachia enzyme from a toxin- antidote like operon is sufficient to induce CI in transgenic fruitflies. Furthermore, the transgenic enzyme is able to reconstitute established cytological phenotypes of CI in the fly. Our results strengthen the “Lock-and-Key” model for CI and support previous observations by contributing a molecular mechanism. The Wolbachia CI system can now be utilized as a transgenic tool for implementation of the sterile insect technique (SIT). This will complement global efforts to fight Zika and vector-borne diseases. Our mechanism also contributes to a broader understanding of how symbionts mediate speciation events in populations.

Abstract #78 Effects of R. Amblyommii Infection on the Vector Competence of Amblyomma Americanum Ticks for R. rickettsii Michael L. Levin* and Alyssa Snellgrove Rickettsial Zoonoses Branch, Centers for Disease Control, Atlanta, GA 30333

Although Dermacentor spp. ticks have long been considered the primary vector of R. rickettsii in the US, other North American tick species are also capable of transmitting the agent, including Rhipicephalus and Amblyomma spp. The lone star tick – A. americanum is aggressive human-biting tick abundant in the south, central, and Mid-Atlantic United States. It have been shown to be a competent vectors of R. rickettsii in laboratory studies. However in nature, A. americanum frequently carry “Rickettsia amblyommii” - another member of the spotted fever group Rickettsia – with the prevalence of infection reaching 84% in some populations. It has been suggested that high prevalence of “R. amblyommii” infection in ticks could affect vector competence of A. americanum for R. rickettsii in nature. We assessed capabilities of R. amblyommii-infected A. americanum larvae to acquire R. rickettsii with a blood meal, transmit it transtadially, and to infect susceptible hosts during nymphal feeding. Larvae transovarially infected with R. amblyommii successfully acquired R. rickettsii from infected guinea pigs. Presence of R. amblyommii did not affect the ability of coinfected engorged larvae to transmit R. rickettsii transstadially. Tree out of 6 guinea pigs infested with coinfected nymphal ticks became PCR- positive for R. rickettsii in the absence of clinical signs of illness, whereas 5 out of 6 became clinically ill when fed upon nymphs infected with R. rickettsii only. None of uninfected larvae placed upon dually infected guinea pigs acquired R. rickettsii with the bloodmeal. Although presence of R. amblyommii in A. americanum does not prevent acquisition and transstadial transmission of R. rickettsii by ticks, the efficiency of horizontal transmission of R. rickettsii by dually infected ticks appears to be significantly diminished.

Abstract #79 Rickettsia parkeri and “Candidatus Rickettsia andeanae” tropism in tissues of experimentally infected Amblyomma maculatum (Gulf Coast tick) Jung Keun Lee*, Gail Moraru, Amanda Harper, John Stokes, Haley Parker, Jacob Hughes, and Andrea Varela-Stokes Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS

American Boutonneuse fever, caused by Rickettsia parkeri, has been recognized in the U.S since 2004. The primary vector of R. parkeri, Amblyomma maculatum, may contain another spotted fever rickettsia of unknown pathogenicity, “Candidatus Rickettsia andeanae”. We investigated tissue tropism of R. parkeri and “Ca. R. andeanae” in singly and co- infected A. maculatum using multiplex QPCR to quantify ratios of rickettsial (ompB) to A. maculatum (MIF) DNA prior to, during, and after blood feeding. Transovarial transmission in ticks and seroconversion in rabbits were also evaluated. Three animal trials were performed using 12 rabbits and capillary-fed adult A. maculatum with cultured rickettsiae in four experimental groups: 1) control; 2) R. parkeri GFPuv; 3) ”Ca. R. andeanae”; 4) R. parkeri GFPuv and “Ca. R. andeanae” combined. Salivary glands, midguts and ovaries (females) of A. maculatum were collected and tested for Days 0 (7 days post-capillary feeding), 6 and 12 after placement on rabbits. QPCR ratios for “Ca. R. andeanae” in salivary glands and midgut of singly and co-infected groups were higher than for R. parkeri in respective groups on Day 0. “Candidatus R. andeanae” was detected at approximately 5-fold higher ratios in salivary glands and midgut tissues on Day 6 than Day 0. 60 Rickettsia parkeri was present at lower ratios than “Ca. R. andeanae” on Day 0 and 6, but was approximately 4-fold higher than “Ca. R. andeanae” in salivary glands and midguts on Day 12 in the co-feeding group. Transovarial transmission of “Ca. R. andeanae” was consistently detected from “Ca. R. andeanae” and co-infected groups, whereas R. parkeri was rarely detected in larvae. Seroconversion of rabbits and detection of “Ca. R. andeanae” in salivary glands supports its transmission. Results from this study provide insight into “Ca. R. andeanae” transmission and the effects of this rickettsia on transmission of the pathogen, R. parkeri.

Abstract #80 The burden of tropical rickettsioses – the Asian perspective Daniel Henry Paris, MD PhD DTMH Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, Oxford University, Oxford, UK

The true burden of tropical rickettsial illnesses remains ill defined. Scrub typhus and murine typhus are the two dominant forms of typhus in the tropics, and typhus represents a leading cause of treatable undifferentiated febrile illness in many regions of Asia. Unfortunately tropical rickettsioses remain under-appreciated and neglected, mainly due to diagnostic difficulties and lack of awareness among medical staff. Although scrub typhus probably is the world’s most important rickettsial illness in terms of disease burden, the available knowledge and literature about Orientia spp. remains limited. It is crucial to improve our understanding of the clinical epidemiology of these illnesses, as these basic data will inform empirical treatment guidelines and diagnostic and treatment algorithms as well as support the discovery and characterization of novel and emerging pathogens of clinical relevance. In this talk the key elements shaping the burden of these diseases will be addressed and limitations discussed; updates on recent causes-of-fever studies across Asia will be presented, and advances in diagnostic methodologies relating to rickettsial illnesses as well as new findings relating to entomology and disease transmission discussed.

Abstract #81 Vascular Permeability with Rickettsial Infections is Abrogated by Membrane-Active Chelators J. Stephen Dumler,1* Valeria Pappas-Brown,1 Emily G. Clemens,1 and Dennis J. Grab2 1Department of Pathology, Uniformed Services University/Walter Reed National Military Medical Center, Joint Pathology Center, Bethesda, MD; 2Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD

The major pathophysiologic derangement in rickettsial infections is increased vascular permeability with hypotension, organ failure, and death. While endothelial cell infection and death should increase vascular permeability, the underlying mechanisms are not well studied, and this could allow development of novel therapeutics to decrease disease severity. Using Trypanosoma brucei rhodesiense, Borrelia burgdorferi, Anaplasma phagocytophilum and Ehrlichia chaffeensis, we noted that divalent cation chelators exert effects on transendothelial electrical resistance (TEER) – an in vitro measure of endothelium barrier permeability. We used real time Electric Cell Impedance Sensing (ECIS) to examine TEER of human brain microvascular endothelial cell (BMEC) barriers with infection by Rickettsia parkeri (spotted fever group rickettsiosis model) and with Ehrlichia chaffeensis (human monocytic ehrlichiosis model). For R. parkeri, we investigated the membrane active chelator (MAC) DPb99 (D-Pharm Ltd.) used in human clinical trials to diminish tissue injury with stroke and pancreatitis. In 4 separate experiments, confluent human BMEC cultures were prepared in electrode array slide chambers to examine TEER with infection. For R. parkeri, cultures with and without DPb99 (0.2, 1, and 5 µg/mL) were also tested. Infection was established using cell-free R. parkeri (MOI of 50:1) in 2 experiments or by incorporating R. parkeri- infected BMEC into monolayers to encompass 5% of cells before MACs. E. chaffeensis was introduced as infected DH82 cells at an MOI of ~10:1. Cultures were monitored for TEER (Ω) changes over ≤80h pi using an Applied Biophysics ECIS Zθ at 4k Hz every 180s. Impedance values were normalized to uninfected, untreated cultures and to baseline values at time 0; changes were evaluated using Student’s t-tests. For E. chaffeensis, TEER decreased 33 and 34% vs. DH82 cells- or medium-only (p=0.020 and p=0.016) at 16 and 19h pi, respectively. R. parkeri decreased TEER within 8 to 24h pi, and this was sustained (40 to 80h). TEER nadirs varied from 90 to 50% of controls among experiments. DPb99 MAC alone had minimal impact on TEER at 0.2 and 1.0 µg/ mL (92-102%), but decreased TEER with 5.0 µg/mL DPb99 after 20-40h to ≥74% of control. Culture with DPb99 significantly reversed decreased TEER with R. parkeri infection compared to no drug 61 controls at 0.2 µg/mL (30h; p=0.039) and 24% at 5 µg/mL (34h; p=0.030). Distinct lineages of Rickettsiales affect endothelial cell barrier integrity in vitro, potentially reflecting major disease mechanisms. Vascular permeability as reflected by TEER is reversed in vitro with MAC drugs safely used in human phase 3 clinical trials. MACs should be studied to better delineate their mechanisms of action with regard to in vitro TEER changes and as potential adjunctive therapies to diminish severe consequences of rickettsial infections and perhaps others where vascular permeability leads to disease. The opinions expressed herein are those of the author(s) and are not necessarily representative of those of the Uniformed Services University of the Health Sciences (USUHS), the Department of Defense (DOD); or, the United States Army, Navy, or Air Force.

Abstract #82 A candidate vaccine induces cellular and humoral immunity and protects from tick-transmitted Ehrlichia chaffeensis infection in a canine host Jodi L. McGill1, Arathy D.S. Nair1, Chuanmin Cheng1, Rachel A. Rusk2, Deborah Jaworski1, Roman R. Ganta1 1Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University; 2Pathobiology Graduate Program, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA

Ehrlichia chaffeensis is the causative agent of human monocytic ehrlichiosis (HME). E. chaffeensis has recently emerged as a frequent cause of severe and fatal tick-borne infection in people in North America. The reservoir host for E. chaffeensis is the white-tailed deer, while humans and dogs are regarded as common incidental hosts. Currently there is no vaccine available for use against E. chaffeensis, and treatment options are limited to a single class of antibiotics. We have recently developed a stable mutant of E. chaffeensis in Ech_0660 gene whose growth is attenuated in vivo in the vertebrate host. We use the canine model of E. chaffeensis infection to demonstrate that vaccination with the Ech_0660 mutant protects from secondary, needle-inoculated or tick-transmitted challenge with wildtype E. chaffeensis. Our results suggest that the Ech_0660 mutant serves as an ideal candidate for vaccine development against E. chaffeensis infectio. The immune responses that correlate with control and clearance of E. chaffeensis, and protection from a secondary challenge, are poorly defined in the physiologic host. We demonstrate that dogs vaccinated with Ech_0660 mutant and challenged with wildtype E. chaffeensis mount a robust CD4 T cell response; with significant production of the Th1 cytokine, IFNγ, and the Th17 cytokine, IL-17. In our study, dogs infected with E. chaffeensis, with or without Ech_0660 mutant vaccination, did not mount a significant CD8 T cell response. Together, our results suggest that the attenuated mutant Ech_0660 represents a promising vaccine candidate for use against E. chaffeensis infection in humans and animals; and that pathogen-specific CD4 T cell responses are likely critical for protection from E. chaffeensis infection in the incidental host.

Abstract #83 An Intradermal Inoculation Mouse Model for Immunological Investigations of Acute Scrub Typhus and Persistent Infection Lynn Soong1,2,*, Nicole L. Mendell1,*, Juan P. Olano1,*, Dedeke Rockx-Brouwer1, Guang Xu1, Yenny Goez-Rivillas1, Claire Drom, Thomas R. Shelite2, Gustavo Valbuena1, David H. Walker1, Donald H. Bouyer1 1Department of Pathology, 2Department of Microbiology and Immunology, School of Medicine, University of Texas Medical Branch, Galveston, TX 77555, USA

Scrub typhus is a neglected tropical disease that puts one-third of the world’s population at risk. This disease is caused by Orientia tsutsugamushi, a Gram-negative bacterium that is transmitted to mammalian hosts during feeding by Leptotrombidium mites and replicates predominantly within endothelial cells. Most studies of scrub typhus in animal models have utilized either intraperitoneal or intravenous inoculation; however, there is limited information on infection by the natural route in mouse skin or its related early host responses. Here, we developed an intradermal inoculation model of scrub typhus and focused on the kinetics of the host responses in the blood and major infected organs. Following ear inoculation with 6 x 104 O. tsutsugamushi, mice developed fever at 11-12 dpi, followed by marked hypothermia and body weight loss at 14-19 dpi. Bacteria in blood and tissues and histopathological changes were detected around 9 dpi and peaked around 14 dpi. Serum cytokine analyses revealed a mixed Th1/Th2 response, with marked elevations of MCP- 62 1/CCL2, MIP- -10 at 9 dpi, followed by increased concentrations of pro-inflammatory markers (IL-6, IL-12, IFN- -CSF, RANTES/CCL5, KC/CCL11, IL- -2, TNF- -CSF), as well as modulatory cytokines (IL-9, IL-13). Cytokine levels in lungs had similar elevation patterns, except for a marked reduction of IL-9. The Orientia 47-kDa gene and infectious bacteria were detected in several organs for up to 84 dpi, indicating persistent infection. This is the first report of an intradermal inoculation model for acute scrub typhus and persistent infection in C57BL/6 mice. This is a significant improvement over current murine models for Orientia infection and will permit detailed studies of host immune responses and infection control interventions.

Abstract #85 Prospective Assessment of the Etiology of Illness after a Tick Bite in Northeastern China Na Jia1*, Xue-Bing Ni1, Jia-Fu Jiang1, Yuan-Chun Zheng2, Qiu-Bo Huo2, Bao-Gui Jiang1, Rui-Ruo Jiang1, Lan-Ma1, Wu-Chun Cao1 1 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, P. R. China; 2 Mudanjiang Forestry Central Hospital, Mudanjiang, 157000, P. R. China

We did a prospective study to understand the etiology of disease associated with tick-bites among patients in Northeastern China, a region of high tick abundance, multiple tick-borne pathogens, and likely human infections. The spectrum of diseases caused by tick-borne pathogens has not been objectively evaluated in this region for clinical management and for comparison with other regions globally where tick-transmitted diseases are common. Approximately forty percentages (75/180) of patients with recent tick-bites were diagnosed with a specific tick-borne disease, including Lyme borreliosis, tick-borne encephalitis, human granulocytic anaplasmosis, human babesiosis and spotted fever group rickettsiosis. When we compared clinical and laboratory features to identify factors that might discriminate tick- transmitted infections from those lacking that evidence, we revealed ultimately that erythema migrans and neurological manifestations were statistically differently presented between those with and without document etiologies (p<0.001, p=0.003). Twelve patients (6.7%, 12/180) were co-infected with two kinds of tick-borne pathogens. Importantly, we demonstrated the poor ability of clinicians to identify the specific tick-borne infectious process that would allow early diagnosis. In addition, it was urgent to develop specific laboratory assays to optimal diagnosis tick-borne diseases.

Abstract #86 Diagnostic Assessment of Testing Blood Verses Serum from Acute RMSF Patients Cecilia Kato*1, Gerardo Alvarez2, Ida Chung1, Christopher Paddock1, Enrique Bolado2 1Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta Georgia; 2Universidad de Sonora, Hermosillo, Mexico

Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever (RMSF) is a severe infectious disease that may have a mortality rate of up to 30% in untreated patients. Clinical diagnosis during the early stages of the disease is often difficult due to the general flu-like syndrome and possible lack of a visible rash. Non-specific early clinical characteristics may include fever, nausea, vomiting, myalgia, anorexia, headache, or maculopapular rash. The potential for a fatal outcome increases if the RMSF patient is not treated as soon as possible or within the first 5 days from symptom onset. The patient might also suffer from permanent organ damage post-recovery due to severe manifestations and/or delayed treatment. Although Rickettsial diagnosis by real-time PCR has been described as a useful tool for the detection of rickettsial DNA in acute patient samples because of the technique’s high sensitivity and specificity, limitations to the accuracy of negative results in blood remain an issue. Because R. rickettsii is an obligate intracellular bacterium it is believed that the number of circulating organisms in blood is minimal during the acute stage of illness. PCR positive samples have been described in both blood and serum from acute patients diagnosed with RMSF. In this study we assess the potential of using serum versus blood for detection of rickettsial DNA in clinical samples. In a retrospective study, 112 samples, representing 56 paired samples from 51 patients were tested with Rickettsia spp. specific assays by standard PCR for the gltA target and by real-time PCR using the PanR8 assay. Overall RMSF diagnosis was confirmed by laboratory testing in 68.6% (35/51) of the patients. A total of 36 paired samples were positive overall. Of these 25 were positive in both blood and serum, 10 positive in blood only, and 1 positive in serum only. 46.4% (25/56) of the samplings would be determined to contain Rickettsial DNA if only serum was assessed verses 62.5% (35/56) if only blood was tested. Therefore

63 from this sampling the preferred samples for RMSF real-time PCR diagnostics are blood and serum or blood alone. Testing of serum alone may lower the accuracy of diagnosis using these methods.

Abstract #87 Theileria buffeli infections in grazing cattle located in mountainous areas of the Republic of Korea Kyoung-Seong Choi1,*, Joon-Seok Chae2, Do-Hyeon Yu3, Jinho Park4, Bae-Keun Park5, Hyeong-Chul Kim6 1College of Ecology and Environmental Science, Kyungpook National University, Sangju 37224, Republic of Korea; 2Laboratory of Veterinary Internal Medicine, BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea; 3College of Veterinary Medicine, Chonnam National University, Gwangju 61186, Republic of Korea; 4College of Veterinary Medicine, Chonbuk National University, Iksan 54596, Republic of Korea; 5College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea; 6College of Veterinary Medicine, Kangwon National University, Chuncheon 24341, Republic of Korea

Objective: Theileriosis is a tick‐borne infectious disease caused by the Theileria species, and transmitted by ixodid ticks of the genus Haemaphysalis [1]. Theileria parasites affect a range of domestic and wild animals, particularly ruminants, and cause economically significant diseases in the livestock industry worldwide. Materials and methods: Whole blood samples were collected from 252 indigenous cattle located in three different regions of the ROK (Hoengseong in the Kangwon province, Jeongeup in the Chonbuk province, and Jeju Island). Historically, the herds from Hoengseong and Jeju Island have grazed on pastureland for at least one season during the warmer months (May‐October) and are housed in stables during the cooler months (November‐April). Results: Sixty‐two samples were verified as positive for Theileria infection by PCR detection methods. Although a high prevalence of Theileria infection was observed in cattle after grazing (29/53 animals, 54.7%), the affected animals exhibited no clinical signs. While the red blood cell (RBC), hematocrit (HCT), and hemoglobin (Hb) values were lower in Theileria‐ positive cattle, anemia was not seen in these animals. Phylogenetic analysis demonstrated that our isolates belonged to the T. buffeli species, phylogenetically related to types A, B, C, and E, with less similarity to T. buffeli type D, which is known to more pathogenic. In addition, T. buffeli identified in Korean indigenous cattle have shown mild to low pathogenicity. Conclusion: These results suggest that the prevalence of T. buffeli is high in the ROK and increases following grazing. Taken together, the prevalence of Theileria infection may not only be seasonally correlated but also may be affected by management practices such as pastureland grazing. Acknowledgements: This work was carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01197804), Rural Development Administration, Republic of Korea.

Abstract #88 Small mammals as hosts of Ehrlichia muris eauclairensis and other rickettsial pathogens in Eau Claire, Wisconsin Bryan N. Ayres*, Audrey E. Osterbind, Jamie L. Perniciaro, Joy A. Hecht, and William L. Nicholson Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, Georgia

A newly emerging pathogen, Ehrlichia muris eauclairensis, has been documented in over 70 human cases since 2009. All patients either resided in or traveled to Minnesota and Wisconsin. This study sought to detect infection of E. muris eauclairensis among small mammals in endemic areas of Wisconsin. Small mammals were trapped in August 2014 and July 2015. Animals were bled, examined for ectoparasites, and necropsied for tissues (lung, spleen, kidney, liver, and ear). Species collected include Blarina brevicauda (1.4%), Clethrionomys gapperi (25.6%), Glaucomys volans (2.3%), Peromyscus leucopus (55.3%), Sorex cinereus (0.9%), Tamias striatus (6.5%), and Zapus hudsonius (7.9%). Samples were screened with a SYBR Green real-time PCR assay designed to detect Anaplasmataceae members. Of the 2014 animals (n=113), at least one organ from 35 animals tested Anaplasmataceae positive. Of the 35 infected animals tested, the three most often infected tissues were kidney (51.4%), followed by blood (37%) and liver (31.4%). Lung and spleen specimens were tested with a real-time PCR assay specific to E. muris eauclairensis and detected two positive P. leucopus (1.8% prevalence). Sequencing of 16S rRNA of two spleen samples indicated a 1.8% (2/113) prevalence with uncharacterized “Candidatus Neoehrlichia spp.” (both from C. gapperi). Anaplasmataceae screening of animals from 2015 (n=102) resulted in 37 animals with at least one positive tissue. Of the 37 infected animals, the three most often infected tissues were ear tissue 64 (70.2%), followed by blood (48.7%) and lung (27%). Anaplasmataceae infected lung and spleen yielded four positive animals for E. muris eauclairensis, one Z. hudsonius and three P. leucopus (3.9% prevalence). Sequencing of the GroESL gene for the remaining infected lung and spleen specimens identified eight additional animals (three P. leucopus and five T. striatus) infected with A. phagocytophilum, while the remaining two animal infections were not yet determined.

Abstract #89 Serological survey of canine tick-borne infections using species-specific serological markers J. Liu1*, M. E. McCown2, C. Fiorello3, D. G. Scorpio4, M. K. Filipovic5, J. Saucier1, B. Thatcher1, R. Chandrashekar1 1IDEXX Laboratories, Inc., Westbrook, ME 04094; 2US Army Veterinary Corps, USA; 3Karen C. Drayer Wildlife Health Center, School of Veterinary Medicine, UC Davis, Davis, CA 95616; 4 Ross University School of Veterinary Medicine, St. Kitts, WI; 5University of Belgrade, Faculty of Veterinary Medicine, Bulevar oslobodjenja 18, 11000 Belgrade, Serbia

Vector-borne diseases (VBD), and more specifically tick-borne diseases, continue to increase over time in global distribution and emergence1. Tick-borne pathogens can cause severe, debilitating diseases in both people and animals, which result in significant medical, work, and production losses2. Dogs have been recognized as sentinels for tick-borne disease surveillance. Sero-surveillance data obtained using conventional serologic tests generally do not provide resolution at species level for tick-borne pathogens. The purpose of this study was to determine the seroprevalence of tick-borne pathogens in canine samples obtained from Serbia, Nicaragua, Colombia and St. Kitts, WI using a research- based species-specific serologic assay Seropositive Rate to Tick-borne Pathogens platform (SNAP® M-A)3. Serum samples were collected as a part of EC ECH EEW APH APL BB routine clinic visit [Serbia, n=156 and Serbia 0.0% 0.0% 0.6% 19.9% 0.6% 1.3% St. Kitts, n=105], field visit [Nicaragua, Nicaragua 0.5% 23.9% 4.3% 1.6% 1.1% 0.0% n=184] and from military working dog Colombia 28.3% 0.0% 0.9% 4.4% 14.2% 0.9% populations [Colombia, n=113]. St. Kitts 30.5% 0.0% 0.0% 0.0% 21.9% 0.0% Serum samples were tested for EC-E. canis; ECH-E. chaffeensis; EEW-E. ewingii; APH-A. phagocytophilum; antibodies to Ehrlichia canis, E. APL-A. platys; BB-B. burgdorferi ewingii, E. chaffeensis, Anaplasma phagocytophilum, A. platys and Borrelia burgdorferi. The serology data are summarized in the Table below. These data indicate exposure to tick-borne pathogens in all sample populations evaluated in this study. While more A. platys and E. canis positives samples were identified from Colombia and St. Kitts, the predominant seropositive samples in Nicaragua were from dogs infected with E. chaffeensis. Additional studies are needed to understand the tick species that transmits E. chaffeensis in Nicaragua. A. phagocytophilum has been reported to be prevalent in some European countries. In Serbia, bordering Central and South Europe, the seroprevalence rate for A. phagocytophilum is as high as 20%. A. phagocytophilum and E. chaffeensis are zoonotic. E. canis infection and infection with an “A. platys-like” organism have also been documented in people. The high levels of exposure to these pathogens revealed in this study warrant precautionary measures for working dogs, working dog handlers, foreign visitors, and the public living and working in these areas. 1Jones et al. Nature. 2008; 451:990-3

Abstract #90 Consistencies in Spatio-Temporal Trends, and Climatic and other Geospatial Risk Factors for Rickettsial Diseases in the Central Midwestern United States Ram K. Raghavan, Gary A. Anderson, Doug. G. Goodin, Roman R. Ganta

The spatial and temporal dynamics of tick populations and the pathogens they vector are highly regulated by fluctuations of climatic and physical environment conditions, and an understanding of such effects on tick-borne disease incidences have public health and economic significance. The spatio-temporal trends and influential factors that affect three tick- borne rickettsial diseases in the Midwestern United States, viz. human monocytic ehrlichiosis (HME), Rocky Mountain spotted fever (RMSF), and bovine anaplasmosis were retrospectively evaluated using case reports submitted to the state health departments and Kansas State Veterinary Diagnostic Laboratory. Robust models that considered space/time influences in addition to those of various climatic and environmental variables were constructed in a Bayesian hierarchical 65 framework. Results indicated that the number of reported cases received on all three diseases have increased over the last decade and were noted to be recorded from previously unreported geographic areas in the region as the years progressed. And, at the spatial scale of counties, poverty level [Odds Ratio (OR) = 2.22, 95% Credible Interval (95% CrI) = 1.82, 2.70), relative humidity (OR = 3.49, 95% CrI = 2.81, 4.33), and an interactive effect between diurnal temperature range and mixed forests (OR = 3.00, 95% CrI = 1.48, 7.12) were identified as significant risk factors for HME. Poverty status (Bayes estimate (BE) = 0.31, 95% CrI = 0.11, 0.41), relative humidity (BE = 0.14, 95% CrI = 0.06, 0.14) and land surface temperature (≥ 35ºC) (BE = -0.31, 95% CrI = -0.07, -0.12) were significant risk factors for RMSF; and, land surface temperature (OR = 0.87, 95% CrI = 0.10, 0.91), relative humidity (OR = 1.53, 95% CrI = 1.15, 2.14) and diurnal temperature range (OR = 0.77, 95% CrI = 0.22, 0.81) significantly affected the spatio-temporal patterns of bovine anaplasmosis. The identification of poverty status as a risk factor for two prominent rickettsial diseases in the region is not preceded by other studies; and, the identification of relative humidity, diurnal temperature range and land surface temperature as important climatological predictors of these diseases is significant in the context of climate-change implications on vector-borne diseases. All three climatological factors are indices of ongoing climate-change and studies have shown shifts in their patterns over the recent decades. How these climatological factors affect the vectors, vector-pathogen and intermediate host-pathogen dynamics is not clearly understood. Further studies evaluating these aspects of the three rickettsial diseases under field and laboratory conditions are therefore needed.

Abstract #91 A distributional survey of ticks (Acari: Ixodida) in Georgia, USA William L. Nicholson Pathogen Biology and Ecology Activity, Rickettsial Zoonoses Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333

The spectrum of rickettsial pathogens found in the United States has grown over the last three decades. Georgia has experienced both increases in rickettsial disease reports and changes in tick occurrence. A meta-analysis of scientific literature was conducted to assess the species and distributional range of ticks across the state of Georgia. This update was needed as the last comprehensive summary of ticks in the state was published in 1972. Literature was searched from 1908 to present for any records of tick occurrence in the state, and any county-level data were extracted. Records were also obtained from museums, natural history collections, and other documented sources. A database was developed to analyze tick species, hosts, and geographical distribution at the county level. Seventeen species of ixodid ticks (representing five genera) and five species of argasid ticks (representing three genera) have been recorded from Georgia. At least one species of tick had been recorded from 133 (83.6%) of the state’s 159 counties. The main human-biting ticks were found in various physiographic regions of the state. Amblyomma americanum was recorded from 72 (45.3%) counties, Dermacentor variabilis from 78 (49.1%) counties, Ixodes scapularis from 79 (49.7%) counties. Previously reported from southern Georgia, Amblyomma maculatum was found in 37 (23.3%) counties. The brown dog tick, Rhipicephalus sanguineus, had been documented from only 17 (10.7%) counties, although wider distribution is suspected. The number of counties with recent tick records was significantly increased over the counties recorded in 1972, and included nine additional counties for Ixodes scapularis not included in a recent update. Field collections will continue to provide additional data and expand our knowledge of ticks in the state. This study will assist public health scientists in understanding landscape risk of exposure to tick species associated with a wide variety of human pathogens.

Abstract #92 Spotted Fever group Rickettsiae in Baltic region Algimantas Paulauskas*, Jana Radzijevskaja, and Asta Aleksandraviciene Department of Biology, Vytautas Magnus University, Vileikos 8, LT‐44404 Kaunas, Lithuania

Tick‐borne rickettsiae are considered to be emerging, but there is still a lack of data on the occurrence and prevalence of the spotted fever group rickettsiae across Europe, especially in the Baltic countries. The first initial investigations of Rickettsia spp. in Lithuania were conducted about forty years ago: Rickettsiae and rickettsia‐like organisms were detected in Ixodes ricinus ticks by a haemocyte test and antibodies against R. sibirica and R. slovaca were found in human and cattle serum samples. However, since that time no other data on rickettsial pathogens or clinical cases due to SFG rickettsiae in 66 Lithuania have been reported. The aim of the present study was to investigate the presence of rickettsiae in sympatric populations of Dermacentor reticulatus and I. ricinus ticks, to characterize the detected rickettsiae using partial sequencing of the gltA gene, and to determine the prevalence of Rickettsia spp. in various regions in Lithuania and Latvia. The molecular analyses of 2490 D. reticulatus and 361 I. ricinus from 56 localities in Lithuania and Latvia revealed the presence of R. raoultii in D. reticulatus and R. helvetica in I. ricinus. In Lithuania, the prevalence of pathogens in D. reticulatus (5.1%) and I. ricinus (17.0%) ranged in different locations from 0% to 37.0% and 0% to 31.3%, respectively. In Latvia, Rickettsia pathogens were found in 4.0% of D. reticulatus and in 20.0% of I. ricinus ticks. Our study is the first demonstration of the presence of R. raoultii in D. reticulatus ticks in the Baltic countries. Our findings provide evidence that the expansion of D. reticulatus ticks to new areas in Baltic countries could increase a risk of tick‐borne spotted fever in this region.

Abstract #93 Spotted Fever Group Rickettsia species identified among ticks in Georgia Ekaterine Zhgenti1*, Roena Sukhiashvili1, Richard J. Obiso3, Christina M. Farris2, Ju Jiang2, Allen L. Richards2 1National Center for Disease Control and Public Health, Tbilisi, Georgia; 2Naval Medical Research Center, Silver Spring, MD, USA; 3Collaborative Biological Research, Tbilisi, Georgia

Introduction: Rickettsia species are obligate intracellular bacteria transmitted via various arthropod vectors to vertebrate hosts. Those Rickettsia spp. transmitted by ticks are mostly classified within the spotted fever group rickettsiae (SFGR) and are often associated with febrile diseases that result in diverse clinical presentations, with severity from mild to severe, and are often characterized by headache, rash, and myalgia. Preliminary studies have shown the presence of SFGR, in particular Rickettsia raoultii, Rickettsia slovaca, and Rickettsia aeschlimannii, among ticks in Georgia. The identity and prevalence of other Rickettsia species in ticks have not been determined. Thus, the objective of this study was to ascertain the identity and prevalence of Rickettsia species among ticks in Georgia. Materials and Methods: A total of 500 single species tick pooled samples (n=1,596 individual ticks) were collected from animals and/or tick drags from eight regions of eastern and western Georgia. Of these, 87 pooled samples (n=442 individual ticks), representing 13 tick species, were identified as Rickettsia-positive by the genus-specific qPCR assay that targets the 17-kDa protein gene. The Rickettsia- positive samples, were subsequently assessed by species-specific qPCR assays for R. raoultii, R. slovaca, and R. aeschlimannii. The tick samples that were positive for Rickettsia but not identified by the species-specific assays were further evaluated by multi-locus sequence typing (MLST) using sequences of three protein-coding genes (gltA, ompA, and ompB). From an initial sample group of 10 tick DNA preparations, we ascertained by MLST that four additional Rickettsia spp. were found infecting the Georgian ticks: Rickettsia massiliae, Rickettsia monacensis, Rickettsia conorii subsp. conorii, and Candidatus Rickettsia barbariae. Species-specific qPCR assays for detection of R. massiliae, R. monacensis, and R. conorii were applied to the unidentified Rickettsia-positive samples. Results: Of the 87 Rickettsia- positive tick pools, 59 samples (67.8%) were identified to the species level by qPCR assays and MLST as: R. raoultii (n=17; 19.5%) from Dermacentor marginatus; R. slovaka (n=12; 13.8%) from D. marginatus, Haemaphysalis sulcata, Ixodes crenulatus, and Rhipicephalus sanguineus; R. aeschlimanii (n=16; 18.4%) from Haemaphysalis punctata, H. sulcata, Hyalomma marginatum, and Hyalomma scupense; R. massiliae (n=2; 2.3%) from H. marginatum; R. monacensis (n=16; 18.4%) from Ixodes ricinus; R. conorii (n=1; 1.1%) from R. sanguineus; and Ca. R. barbariae (n=2; 2.3%) from Rhipicephalus annulatus. Five tick pools were positive for both R. raoultii and R. slovaca (5.7%) and one pool for R. aeschlimanii and R. massiliae (1.3%). Twenty-two (25.3%) of the Rickettsia-positive samples haven’t been identified by the aforementioned species-specific qPCR assays and require further MLST analysis. Conclusion: The detection and identification of the SFGR species R. massiliae, R. monacensis, R. conorii, and Candidatus R. barbariae for the first time were made from tick samples in Georgia. This information plus the prevalence and distribution of Rickettsia species among ticks from different regions of Georgia is extremely valuable to health care providers, residents and travelers to the newly identified endemic region for tick-borne rickettsial pathogens to prevent or reduce the risk of rickettsial diseases.

67 Abstract #94 Risk assessment for vector-borne diseases at Gateway National Recreation Area, NY: monitoring ticks. Preliminary study, 2014 Ellen Stromdahl*1, Garrett Heck1, Mary Vince1, Waheed Bajwa2, Zahir Shah2, David Wong3 1Army Public Health Center, Aberdeen Proving Ground, MD; 2New York City Department of Health and Mental Hygiene, New York, NY; 3National Park Service Office of Public Health, Albuquerque, NM

Autochthonous cases of tick-borne illness (babesiosis, human granulocytic anaplasmosis, human monocytic ehrlichiosis, Lyme disease, Rocky Mountain spotted fever) have been recently reported from the boroughs of New York City (Brooklyn, The Bronx, Manhattan, Staten Island and Queens) to the New York City Department of Health and Mental Hygiene. The species distribution and human pathogen prevalence of ticks has been reported from some regions of the state of New York, but there have been no investigations of ticks in New York City. The most accurate way to assess the risk of tick- borne disease is surveillance of infected ticks, therefore, in 2014, at Gateway National Recreation Area, NY (GATE), a collaborative tick collection study was initiated involving the US National Park Service (NPS) Office of Public Health, the New York City Department of Health and Mental Hygiene (NYC DOHMH), and the Army Public Health Center (APHC). Host- seeking ticks were collected by NYC DOHMH and NPS personnel at monthly intervals from 24 April - 25 November, 2014. These were analyzed for human pathogens using PCR at the APHC Tick-Borne Disease Laboratory. A total of 1,160 ticks were collected: 107 Amblyomma americanum, 874 Dermacentor variabilis, 16 Haemaphysalis leporispalustris and 163 Ixodes scapularis. PCR of I. scapularis revealed Anaplasma phagocytophilum sequences in 3/116 (2.6%) nymphs, Babesia microti sequences in 2/47(4.3%) adults and 4/116 (3.5%) nymphs, and Borrelia burgdorferi sequences in 17/47(36%) adults and 31/116 (27%) nymphs. Three of the four I. scapularis nymphs (3/116 = 2.6%) infected with Ba. microti were also infected with B. burgdorferi. PCR of A. americanum revealed Ehrlichia chaffeensis sequences in one nymph (1/97 = 1%), Ehrlichia sp. “Panola Mountain” in another nymph (1/97 = 1%), and Rickettsia spp. sequences in 8/10 (80%) adults and 44/97 (45%) nymphs. PCR of D. variabilis revealed Rickettsia spp. sequences in 15/874 (1.7%) adults. Further work will include speciation of Rickettsia, mapping tick species distribution and pathogen prevalence by GATE site, and analysis of ticks collected in 2015. The study will continue through 2017.

Abstract #95 Evaluating an Ecological Niche Model for Rickettsia montanensis infected Dermacentor variabilis based on ticks collected from humans using environmentally captured ticks. Heidi K. St. John1,2, Penny M. Masuoka2, 3, Melissa K. Miller5, Melissa L. Adams1,2, Johanna G. Flyer-Adams1, Ju Jiang1,2, Patrick J. Rozmajzl1, Ellen Y. Stromdahl4, and Allen L. Richards 1,3 1 Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; 2 Henry M. Jackson Foundation, Bethesda, Maryland; 3 Department of Preventive Medicine and Biostatistics, Uniformed Services University of the Health Sciences, Bethesda, Maryland; 4 Laboratory Sciences, Army Public Health Center, Aberdeen Proving Ground, Maryland; 5 Laboratory Sciences, Army Public Health Center, Fort Meade, Maryland

The tick-borne spotted fever group of rickettsia (SFGR), Rickettsia montanensis, has been considered a non-pathogen until a potential human infection was reported in 2012. With this in mind the prevelance, distribution, and potential risk of R. montanensis in Dermacentor variabilis was revisted using ticks obtained from Department of Defense (DoD) personnel and their dependents at DoD Medical Treatment Facilities (MTF) during 2002 – 2012. The risk potential was estimated based on the ecological niche models (ENMs) of Rickettsia montanensis-positive and –negative Dermacentor variabilis. ENMs need to be tested with additional data to improve or confirm an existing model. This study used environmentally collected D. variabilis tick data obtained from the Public Health Command Atlantic (Provisional) and West Virginia Department of Health and Human Resources to test our existing model. The environmentally collected ticks when incorporated into the existing model, yielded ENMs that were influenced by the same variabled, but differed slightly from the original models. The ENMs developed in this study were confirmatory and could benefit future R. montanensis pathogencity studies, however inclusion of more geographically diverse locations would be beneficial.

68 Abstract #96 Survey for Rickettsia species in Dermacentor variabilis ticks collected from National Parks and Monuments across the Eastern United States Joy A. Hecht1, Tammi L. Johnson2, Rebecca J. Eisen2, Christopher D. Paddock1, Sandor E. Karpathy1* 1Rickettsial Zoonoses Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Diseases, Centers for Disease Control and Prevention; 2Bacterial Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Diseases, Centers for Disease Control and Prevention

Dermacentor variabilis, a human-biting tick found throughout the eastern half of the United States, is a vector of multiple human pathogens, including Francisella tularensis and various rickettsial species. Historically, D. variabilis has been considered the primary vector of Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever. We screened 202 D. variabilis ticks collected during 2014 and 2015 from 7 different national parks located in 5 states in the eastern United States for the presence of rickettsial DNA. Tick extracts were evaluated using three different real-time assays, including one specific for R. rickettsii, one specific for Rickettsia bellii and one genus-specific assay. Samples negative for R. bellii and R. rickettsii, but positive by the pan-rickettsial real-time assay were screened using a nested ompA PCR assay, and amplicons were sequenced to determine the identity of the Rickettsia species. We identified 2 (1%) ticks positive for R. bellii and found no R. rickettsii positive samples. Of the 202 ticks screened, 50 (25%) were positive for a Rickettsia species, including 7 (3%) confirmed as Rickettsia montanensis using DNA sequencing. Further sequencing analysis is required to identify other species of Rickettsia present.

Abstract #97 Flea-borne Rickettsiae in Southeastern Georgia, USA Marina E. Eremeeva1*, Danielle Capps1, Amanda Jo Williams-Newkirk2, Gregory A. Dasch3, Lorenza Beati4, Lance Durden4. 1Jiann-Ping Hsu College of Public Health, Georgia Southern University, Statesboro, GA; 2Enteric Diseases Laboratory Branch, CDC, Atlanta, GA; 3Rickettsial Zoonoses Branch, CDC, Atlanta, Georgia; 4College of Science and Mathematics, Georgia Southern University, Statesboro, GA.

Background: Southeastern Georgia was historically known as an endemic zone for murine typhus, a flea-borne disease caused by Rickettsia typhi. Successful rodent and flea control programs lead to eradication of the disease, so its actual occurrence in the area is unknown while reports of worldwide murine typhus re-emergence are common in the literature. To address this existing information gap, our study was conducted to characterize the rickettsiae that are associated with fleas infesting companion and wild animals in Southeastern Georgia. Methods: Fleas were collected from companion cats, dogs, and from opossums and a cotton rat that were trapped in woodland nonhuman inhabited areas in Bulloch, Fulton, Jefferson and Warren Counties, Georgia in 2011-2014. Archival flea samples collected from opossums in 1992- 2010 were also included. The fleas were identified to species and the DNA was extracted using Qiagen DNeasy kit according to the manufacturer’s directions. Flea DNA were individually tested using TaqMan assays specific for R. typhi ompB and R. felis bioB followed by sequencing of gltA fragment. Results: In total, 387 fleas were tested. Only Ctenocephalides felis (n=68) were collected from cats. Dogs were infested with C. felis (n=13) and Pulex simulans (n=2). Live-trapped opossums were mainly infested with Polygenis gwyni (n=68) while only a few C. felis (7) and Orchopeas howardi (3), were found on this host. C. felis was predominant species among archival samples. R. typhi DNA was detected by ompB TaqMan in 9.3% of fleas tested, including 5.6% of P. gwyni and 13% of C. felis tested. R. felis DNA was detected in 56.14% of all fleas, including 76.61% of C. felis and 14% of P. gwyni tested. Sequencing of the Rickettsia gltA fragment provided evidence that Rickettsia asemboensis is co-circulating in the same populations of fleas. Summary and Conclusions: We demonstrated that R. typhi is still present in Georgia, USA but there is a predominant circulation of R. felis in C. felis in Georgia. Furthermore, we identified R. asemboensis in the same region. Since these three flea-borne rickettsiae have different pathogenic potential, the availability of more accurate differential diagnostic assays is critical for accurate identification of the flea-borne rickettsioses in patients in Georgia and their proper clinical management. Co- infections with more than one flea-borne agent cannot be excluded

69 Abstract #98 Prevalence of Q fever and Rickettsia in ticks in Northeastern Azerbaijan Rita Ismayilova*, Agarahim Mammadsalahov, Rakif Abdullayev, Esmiralda Seyidova, Sabina Ibrahimova, and Elvin Naghiyev Republican Anti-Plague Station, Baku, Azerbaijan

Rickettsial diseases and Q fever are encountered all over the world. Coxiella burnetii, the pathogen causing Q fever, is similar to other rickettsia organisms, but is classified in its own genus. Research was conducted in the northeastern region of Azerbaijan. A total of 40 villages in 3 regions (17 villages in Khachmaz, 14 villages in Guba, and 9 villages in Gusar) were selected for the study. Ticks from cattle, small ruminants, and backyard poultry (including chickens, ducks and geese) were collected manually. Ticks were sorted into different tubes and labeled individually (name of the village, kind of animal collected from, GPS longitude and latitude) for identification and nucleic acid extraction. Tick species were identified in the laboratory. Samples designated for laboratory analyses were pooled by species (regardless of the life phase) from a given village. The quantity of arthropods in each sample depended on the number of the same type of arthropods collected from the same area and ranged from 1 to 10 ticks per pool. The nucleic acids of the grouped and homogenized vectors were extracted using Qiagen QIAamp mini kits for RNA extraction (Valencia, CA, USA). Tick samples were prepared for laboratory diagnostics using real-time PCR. Out of the 3,578 ticks collected, 682 pools were created. Eight pools were positive for Q fever (1.2%) and 58 pools were positive for Rickettsia (8.5%). Q fever nucleic acid was detected in Rhipicephalus (Boophilus) annulatus, Dermacentor marginatus, and Rhipicephalus sanguineus tick species. Rickettsia nucleic acid was detected in Rhipicephalus annulatus, Dermacentor marginatus, Rhipicephalus sanguineus, Argas persicus, Ixodes ricinus, Hyalomma plumbeum, and Hyalomma asiaticum species. This research enabled a first-time study of Q fever prevalence in ticks in the northeastern part of Azerbaijan. The results will assist the public health system in researching the potential risk factors for arthropod-led infections, as well as in strengthening epidemiological control of these diseases.

Abstract #99 Coxiella burnetii small RNA 12 (CbsR12) targets carA transcripts in vitro Shaun Wachter*, Indu Warrier and Michael F. Minnick Division of Biological Sciences, University of Montana, Missoula, Montana

Coxiella burnetii is an obligate intracellular bacterial pathogen and the etiological agent of Q fever. Previous transcriptomic analyses by our group revealed several novel C. burnetii small RNAs of varying sizes and expression patterns. Sequence alignments of the sRNA’s across all strains of C. burnetii showed strong conservation, suggesting a functional role for these RNA’s in Coxiella’s intracellular lifestyle. Furthermore, due to C. burnetii’s biphasic life cycle of metabolically active (LCV) and inactive (SCV) states, several of sRNA’s were shown to be differentially expressed in SCV and LCV cells by RNA-Seq and Northern blot analyses. One such sRNA, termed CbsR12, showed a marked upregulation in SCV’s when compared to LCV’s, in both axenic media and infected tissue cultures. To identify potential targets for CbsR12, three separate in silico sRNA target prediction programs were employed, and targets common to all three were chosen for further study. RNA- RNA electrophoretic mobility shift assays (EMSAs) of in vitro-transcribed CbsR12 and the potential in silico targets has revealed CbsR12 binding to carA transcript, whose respective gene codes for carbamoyl-phosphate synthase subunit A. CarA catalyzes the first step in pyrimidine biosynthesis and has been implicated as an effector that counters nitrosative stress within the Coxiella-containing vacuole (CCV). Since neither of these CarA-associated functions would likely be beneficial to the SCV, we hypothesize that carA is down-regulated by Cbsr12. A knockdown of CbsR12 utilizing a CRISPR/dCas9 system is currently being developed in order to determine the fitness advantage of CbsR12 in vivo and to assay its effects on carA expression in SCV and LCV cells.

70 Abstract #100 Alterations in Dermacentor andersoni transcription in response to Anaplasma marginale infection Forgivemore Magunda1,2, Sebastian Aguilar1, Kelly A. Brayton1,2, Susan M. Noh1,2,3* 1Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA; 2The Paul G. Allen School for Global Animal Health, Washington State University, Pullman WA; 3Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, WA

Ticks are vectors of many important diseases of livestock and humans including those in the family Anaplasmataceae. Successful ongoing transmission of these pathogens relies on the ability to invade and replicate within the midgut epithelial cells. The midgut is functionally unique as the blood meal is digested by a complex network of vacuolar peptidases. Anaplasma marginale modulates the intravacuolar environment to prevent acidification and accumulation of peptidases. Corresponding global alterations of the midgut that allow for pathogen colonization are unknown for Dermacentor andersoni, a natural vector of A. marginale. In the current study we used Ion torrent high throughput RNA sequencing to compare the midgut transcripts of unfed D. andersoni male ticks to those fed on either an uninfected or A. marginale-infected animal. Importantly, 100% of ticks in the A. marginale-exposed group were infected with an average 4x105 +/-0.83 A. marginale per midgut. After sequencing and assembly, reads from all groups were pooled, and following data clean up, 11,716 transcripts were annotated. Reads corresponding to each study group were analyzed using K-means clustering. Several clusters were highly differentially transcribed. Most remarkable were four genes, of unknown function, that were highly up-regulated in response to A. marginale infection. These findings are similar to those in Rhipicephalus microplus, a vector of A. marginale common in many tropical regions as reported by Mercado-Curiel et al., (2011) in which few genes, primarily annotated as hypothetical or of unknown function, were up-regulated in response to A. marginale infection in the midgut. Understanding the function of these genes will help to determine the molecular foundation of vector competence.

Abstract #101 Characterization of Small, Noncoding RNAs in Rickettsia prowazekii Casey L. C. Schroeder1*, Hema P. Narra1, Mark Rojas2, Abha Sahni1, Kamil Khanipov2, Jignesh Patel1, Yuriy Fofanov2, and Sanjeev K. Sahni1 1Department of Pathology, The University of Texas Medical Branch, Galveston, TX 2Department of Pharmacology, The University of Texas Medical Branch, Galveston, TX

Bacterial small RNAs (sRNAs), originating from either intergenic regions within the genome (trans-acting) or the antisense strand of an open reading frame (cis-acting), are now appreciated as highly important post-transcriptional regulators of bacterial virulence and growth genes, but the knowledge of their existence, characteristics, and potential roles in the lifecycle of pathogenic Rickettsia species remains in its infancy. We have recently reported on a comprehensive bioinformatics-based analysis of sRNAs in Genus Rickettsia, documenting the presence of 12 sRNAs in R. prowazekii, the causative agent of epidemic and recrudescent typhus in humans. Application of RNA sequencing for in-depth detection of R. prowazekii sRNAs expressed during the infection of human microvascular endothelial cells (HMECs), which represent the primary host targets during human infections, revealed the presence of 35 and 23 trans- and cis-acting sRNAs dispersed throughout the genome, respectively. Along with the previously identified 12 sRNAs, these newly identified sRNAs were named as Rp_sR1 through Rp_sR70. Using strand-specific probes for Northern hybridization, we confirmed the expression of four well-known sRNAs (6S RNA, α-tmRNA, RNaseP_bact_a, and 4.5S RNA) and 3 novel rickettsial sRNAs (Rp_sR17, Rp_sR47, and Rp_sR60) in R. prowazekii-infected HMECs. Transcription start sites (TSSs) for Rp_sR17, Rp_sR34, Rp_sR47, Rp_sR60, and Rp_sR67 were next determined using 5’-RNA Ligase Mediated-Rapid Amplification of cDNA Ends and found to be corresponding to the read coverage observed in RNA-seq data, providing evidence for independent genesis of these sRNAs. Next, strong σ70 promoters were also identified in the region upstream of TSSs indicating independent transcription. Predicted secondary structures using RNAfold revealed characteristic sRNA features, such as the presence of arms, bulges, and stem loops, further authenticating rickettsial sRNAs. Combined, these results clearly demonstrate expression of R. prowazekii sRNAs in host cells and unravel new insights into the organization of its genome and potential mechanisms underlying the regulation of its transcriptome.

71 Abstract #102 Metagenomics of Head Lice Using the Ion Torrent PGM Gregory A. Dasch1*, Zachary C. Holmes1, T. Brian Shirey2, Marina E. Eremeeva3, and Sarah Zohdy4 1Rickettsial Zoonoses Branch, CDC, Atlanta, GA 30329; 2Enteric Diseases Laboratory Branch, CDC, Atlanta, GA 30329; 3Jiann-Ping Hsu College of Public Health, Georgia Southern University, Statesboro, GA 30460; 4School of Forestry & Wildlife Sciences, Auburn University, Auburn, AL 36849

The body louse, Pediculus humanus corporis, is the vector of the etiologic agents of three important public health diseases, Rickettsia prowazekii (epidemic typhus), Bartonella quintana (trench fever), and Borrelia recurrentis (louse-borne relapsing fever). While the head louse P. humanus capitis, has vectorial capacity for R. prowazekii, and B. quintana has been detected in it sporadically, the genetic distinction between different stocks of these two lice has been controversial. Recent complete genome sequencing (Illumina HiSeq) of the complex 18 minicircles of the mitochondria of head and body lice from Australia, Ethiopia, Nepal, Iran, China, and France and the rabbit maintained Orlando body louse strain from the USA permitted identification of 19 clades of lice while partial sequences of cox1 and cytB identified only 3 clades. We sequenced three shotgun libraries on the Ion Torrent PGM (318v2 chips with 200 bp chemistry) to determine the metagenome of Qiagen spin column extracted DNA following whole gene amplification (WGA) with the REPLI-G mini kit (Qiagen) of DNA from two female head lice from Madagascar and DNA without WGA from a nymph from Georgia, USA. Sequences were read mapped with Geneious to the sequences from the head louse mitochondrial minicircles (Shao et al. 2012), the Pediculus body louse (Orlando) and its Riesia endosymbiont chromosome and plasmid pPan (Kirkness et al. 2010). Although louse and Riesia DNA were detected in all three samples, the relative amounts of sequences mapping varied dramatically for each sample (0.002-7.38%). The nymph had the best Riesia chromosome coverage and one of the WGA females had the best pPan coverage. SNPs compared to the body lice sequences were obtained in both cases. WGA increased the average percentage of minichromosome reads from 0.78% (range 0.29-1.76%) obtained for the nymph to 3.41 (range 1.46-9.53%) and 2.74% (range 1.16-7.02%). All three samples gave consensus sequences within a few bases of the expected sizes of all 18 minicircles and sufficient depth to identify SNPs. Each of the mitochondrial minicircles of the two Madagascar female lice were very similar in sequence and could be differentiated from those of the Georgia louse nymph; both were related to clades containing the most common head and body lice genotypes from France, Africa and Australia. The Ion Torrent PGM provides excellent mitochondrial minichromosome DNA sequences from both WGA amplified and unamplified DNA from lice at a relatively low cost and can provide some useful data on its endosymbiont Riesia.

Abstract #103 Decoding the post-transcriptional regulatory networks in Rickettsia conorii Hema P. Narra1*, Casey L.C. Schroeder1, Abha Sahni1, Yuriy Fofanov2, and Sanjeev K. Sahni1 1Department of Pathology and Institute for Human Infections and Immunity, The University of Texas Medical Branch, Galveston, TX; 2Department of Pharmacology, The University of Texas Medical Branch, Galveston, TX

Mediterranean spotted fever caused by Rickettsia conorii continues to present a significant health threat to individuals belonging to all age groups. The regulatory mechanisms enabling R. conorii persistence in the arthropod vector and switch to a virulent phenotype causing significant morbidity/mortality upon infection of human hosts have remained elusive. We have determined the transcriptional landscape of R. conorii during the infection of Human Microvascular Endothelial Cells (HMECs) and identified 17 trans-acting (intergenic) and 22 cis-acting (antisense) novel small RNAs (hereafter referred as ‘Rc_sR’) employing combinatorial approaches of bioinformatics and RNA sequencing. Independent expression of three novel trans-acting sRNAs (Rc_sR33, Rc_sR35 and Rc_sR42) in addition to known bacterial sRNAs (ssrS, ssrA, ffs and RNaseP_bact_a) was next confirmed by Northern hybridization. To conduct a comparative analysis of the expression profile of novel trans-acting sRNAs, we infected HMECs and Amblyomma americanum tick (AAE2) cells with R. conorii in vitro and C3H/HeN mice in vivo. Interestingly, Rc_sR35 and Rc_sR42 showed significantly higher expression in HMECs as compared to tick cells, whereas Rc_sR31 and Rc_sR33 showed similar levels of expression in both cell types. Most intriguingly, Rc_sR31 was significantly upregulated in the lungs and brain, but not testes, of infected mice. We have predicted 680 target genes potentially involved in 303 biological processes to be regulated by trans-acting sRNAs and

72 experimentally validated the interaction of Rc_sR31 with RC0877 (putative ankyrin repeat protein) and Rc_sR42 with cydB (cytochrome bd-I ubiquinol oxidase subunit II) by electrophoretic mobility shift assay. In sum, our study is the first to convincingly demonstrate the existence of post-transcriptional riboregulatory mechanisms in R. conorii. Further studies on differential regulation of rickettsial sRNAs and transcriptome during the infection of mammalian vis-à-vis tick vector host cells in vitro and target organ systems in vivo are now in progress.

Abstract #104 Cytoskeleton interactions with the Coxiella burnetii parasitophorous vacuole Heather Miller*, Charlie Larson, and Robert Heinzen Laboratory of Bacteriology, Rocky Mountain Laboratories, Hamilton, MT

Coxiella burneti, the cause of Q fever, is an intracellular bacterium that ex vivo can invade and replicate within a variety of phagocytic and non-phagocytic cell types. Upon entry into a host cell, C. burnetii traffics through the endosomal pathway, eventually residing in a phagolysosome. Here C. burnetii establishes a modified parasitophorous vacuole (PV) in which to replicate by promoting fusion with intracellular vesicles. The mature PV can expand to occupy the majority of the host cell cytoplasm. How the structural integrity of the large and spacious PV is maintained within the host cell is unexplored. Chlamydia trachomatis and Salmonella typhimurium exploit host cytoskeletal components to support their intracellular vacuoles. Moreover, filamentous actin (F-actin) is reported to surround the C. burnetii PV in Hela epithelial cells and THP- 1 macrophages. The biochemical roles of F-actin and other cytoskeletal elements in PV function are unclear. Here we employed confocal microscopy to identify PV interactions with F-actin, microtubules and intermediate filaments (IF). F- actin localized to the PV, but unlike previous reports, appeared in a punctate pattern around the vacuole. Microtubules were present around the PV, but lacked a clear association. Intermediate filaments comprised of vimentin, cytokeratin 8, or cytokeratin 18 enveloped the mature PV, with vimentin localization the most striking. Vimentin disassociated from the PV in cells treated with the IF inhibitor, withaferin A, then reassembled upon inhibitor removal, suggesting a regulated interaction between the PV and vimentin. Interestingly, C. burnetii formed normal looking PVs in cell lines lacking vimentin that did not show enhanced sensitivity to osmotic disruption. Although we have not demonstrated an obvious role for the cytoskeleton in PV stability and maintenance, it is of interest to determine the function of PV-associated IFs and F-actin, as besides affording structural support, these filaments can mediate vesicle transport and intracellular signaling.

Abstract #105 Examination of tick vectorial capacity factors during tick-borne bacterial infection Kathryn E. Reif1*, Jessica K. Ujzco2, Deb C. Alprin2 and Susan M. Noh2 1Center for Excellence in Vector Borne Diseases, Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, Kansas 2Animal Disease Research Unit, Agriculture Research Service, US Department of Agriculture, Pullman, Washington

Changes in environmental conditions and increased human and animal movement have influenced tick-borne pathogen epidemiology, including diversification of vector species. Identifying the factors that drive vector competence and more globally vectorial capacity are essential in understanding tick-borne disease ecology and designing effective intervention strategies. Vectorial capacity of ticks for tick-borne pathogens is shaped by both intrinsic and extrinsic factors. Extrinsic factors include environmental variables such as temperature, humidity, nutrition and population density. Related more directly to vector competence, intrinsic factors affect the ability of the tick to acquire and transmit a given pathogen. Intrinsic factors include physical tissue barriers, immune pressure, and co-occurring microbe interactions within the tick. We are broadly interested in determining molecular mechanisms underlying vector competence for tick-borne bacterial pathogens. Towards this goal, we evaluated vector competence-related factors during Francisella tularensis infection of Dermacentor andersoni and Ixodes scapularis, an ecologically significant and insignificant vector of tularemia, respectively. Using F. tularensis subsp. novicida (F. novicida) and tick cell lines derived from D. andersoni (DAE100) and I. scapularis (ISE6), we examined if inherent vector competence differences underpinned observed ecological differences. Specifically, we investigated whether there were differences in F. novicida i) invasion, ii) replication, or iii) effect on tick cell viability between DAE100 and ISE6 cells. Both tick cell lines were permissive to F. novicida infection; however, infection kinetics differed with significantly greater bacterial infection levels observed in DAE100 compared to ISE6 cells where infection 73 frequently fell below detection limits. Significantly greater host cell mortality was observed in DAE100 cells compared to ISE6 cells over the course of infection which was directly reflective of the reduced bacterial levels in ISE6 compared to DAE100 cells. Because temperature shifts occur after tick feeding, we also examined whether temperature influenced F. novicida infection kinetics in tick cells. Compared to 34°C, decreasing assay temperature to 24°C resulted in prolonged high bacterial burdens in both cell lines and significantly reduced DAE100 cell mortality. Tick-borne pathogens are well adapted to the tick vector with transmission dependent on the pairing of competent vectors and pathogen genotypes. The results reported here reflect this relationship with greater sustained Francisella infection rates observed in the more ecologically relevant tick species at environmental temperatures. Although cell culture systems cannot recapitulate the myriad factors that determine vector competence in vivo, they can be useful when examining the influence of specific variables such as vector species and temperature. These assays provide a foundation for future in vitro and in vivo studies to examine specific molecular mechanisms that drive vector competence and tick-borne pathogen vector tropism.

Abstract #106 Nuclear trafficking of the anti-apoptotic Coxiella burnetii effector protein AnkG requires binding to p32 and Importin-α1 Walter Schäfer1, Rita A. Eckart1, Benedikt Schmid2, Kerstin Hof2, Yves Muller2, Bushra Amin3, and Anja Lührmann1* 1Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany; 2Lehrstuhl für Biotechnik, Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, D-91052 Erlangen, Germany; 3Lehrstuhl für Biochemie, Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, D 91058 Erlangen, Germany;

The obligate intracellular bacterium Coxiella burnetii is the causative agent of the zoonotic disease Q-fever. Q-fever is often a mild flu-like illness, but can develop into an atypical pneumonia or hepatitis. Furthermore, the infection can lead to chronic infection which is typically characterized by bacterial endocarditis and is potentially fatal. C. burnetii pathogenesis depends on a functional type IVB secretion system (T4BSS), used to translocate bacterial proteins into the host cell in order to manipulate host cell pathways. To date over 130 effector proteins have been identified, however their functions mainly remains elusive. We have demonstrated that the T4BSS effector AnkG inhibits host cell apoptosis. Binding of AnkG to the host cell protein p32 is crucial for migration of AnkG into the nucleus and nuclear localization of AnkG is essential for its anti-apoptotic activity. However, the mode of action of AnkG is still not fully understood. Here, we identified Importin-α1 as an additional host cell binding partner of AnkG and could show that the amino acid 11 of AnkG is important for this interaction, but not the binding to p32. Importin-α1 is an adaptor protein that links cargo proteins to a carrier protein that actually takes the cargo through the nuclear pore complex. Trafficking of AnkG into the nucleus and thus anti-apoptotic activity depends on binding to Importin-α1. Most importantly, our results demonstrate that during C. burnetii infection translocated AnkG localizes within the host cell nucleus and that this localization depends on binding to p32 and Importin-α1. Taken together, the anti-apoptotic activity of AnkG is controlled by p32-mediated intracellular trafficking and Importin-α1 mediated nuclear entry.

Abstract #107 Effects of novel bacteriophage on Abalone exposed to Candidatus Xenohaliotis californiensis – the causative agent of Abalone Withering Syndrome. Ashley Vater1, Barbara A. Byrne2, James D. Moore3* 1Integrative Pathobiology Graduate Group, UC Davis, Davis CA 95616; 2Pathology, Microbiology, Immunology, UC Davis, Davis CA 95616; 3Shellfish Health Laboratory, California Department of Fish and Wildlife, Bodega Bay, CA 94923

A Rickettsiales-like pathogen - Candidatus Xenohaliotis californiensis – has decimated abalone populations along the Pacific coast of North America. White Abalone, – Haliotis sorenseni – are particularly susceptible and are now nearly extinct in the wild. The species is federally endangered, with a small number of animals present in a captive breeding program centered at the UC Davis Bodega Marine Laboratory. Ca. X. californiensis proliferates in epithelial cells of the abalone gastrointestinal tract and causes clinical signs of starvation. In 2012, evidence of a putative phage associated with Ca. X. californiensis was published. A subsequent study supported a phage-associated reduction of the bacteria’s pathogenicity in black abalone. The current project investigated the effects of phage in white and red abalone species under temperature 74 regimes that either slow (cold water) or enhance (warm water) disease progression. Using a comparative experimental design, we observed differences over time in mortality and growth rates of the two species and bacterial load by qPCR. By day 215, 92% of white abalone exposed to the Ca. X. californiensis and its phage in the warm water (18°C) treatment died. However, red abalone exposed to the same conditions faired far better, with a mortality rate of 10%. In comparison to experiments conducted prior to the presence of the phage, red abalone show a 40% survival rate improvement in the presence of phage. These data further support the phage as a protective and possibly therapeutic agent in red abalone while white abalone remain susceptible to withering syndrome in its presence. These findings have important implications for implementation of a white abalone recovery program, particularly with respect to the thermal regimes of locations where captively reared individuals will be outplanted.

Abstract #108 Ehrlichia chaffeensis TRP120 Effector Autoubiquitinates and Contains a C-terminal HECT E3 Ligase Domain Bing Zhu1*, Tierra R. Farris2, Seema Das1, Jere W. McBride1-5 1Department of Pathology and 2Microbiology & Immunology, 3Center for Biodefense and Emerging Infectious Diseases, 4Sealy Center for Vaccine Development, 5Institute for Human Infections and Immunity, Univ. of Texas Medical Branch

Ehrlichia chaffeensis modulates numerous host cell processes, including gene transcription to promote intracellular survival in the mononuclear phagocyte. E. chaffeensis TRP120 is a multifunctional effector involved in ehrlichial entry and is a nucleomodulin. We previously reported TRP120 is conjugated with SUMO2/3 isoforms in a C-terminal canonical SUMO motif, which mediates interactions with host proteins. Inhibition of the SUMO pathway also decreases ehrlichial replication and intracellular survival. However, little is known about other potential post translational modifications (PTMs) of the TRP120 effector and the functional implications. In this study, we demonstrate that TRP120 is conjugated by ubiquitin (Ub). E. chaffeensis infected cell lysates were subjected to ubiquitin enrichment, and multiple TRP120 isoforms ranging in mass were identified, demonstrating multi-ubiquitination of native TRP120. The C-terminus of TRP120 exhibited significant sequence similarity (~50%) with several known HECT E3 ligases. TRP120 contains cysteine residues in the C terminus at positions 441 and 494, which are consistent with the catalytic cysteine site in HECT E3s. Next, we examined the ubiquitination of TRP120 in vitro using recombinant protein in presence of E1 and different E2 enzymes. We observed that TRP120 can autoubiquitinate in presence of Ubch5b, Ub, ATP, Mg2+. This suggests that TRP120 has E3 ubiquitin-protein ligase activity. In addition, using immunofluorscence microscopy, co-IP and an in vitro ubiquitination assay, we found that the HECT E3 ubiquitin ligase Nedd4L colocalizes with TRP120 at ehrlichial inclusions during E. chaffeensis and also facilitates TRP120 ubiquitination. Nedd4L knockdown resulted in the accumulation of unmodified TRP120. To further identify the ubiquitination sites on TRP120, a microarray on microfluidic chip containing TRP120 peptides was incubated with the E1 and cell lysate. Positive signals were detected at the site corresponding to K120, K396 in tandem repeat region and K432 in C-terminal, while we found null signal at mutant site where the target lysine was replaced with arginine(R). Future directions for this study include identification of host substrates targeted by TRP120, elucidation of the role of TRP120 ubiquitination in infection, and further reveal a novel ubiquitin pathway by which E. chaffeensis exploits the host cell PTM to promote survival.

Abstract #109 Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Directly Regulates Host Gene Expression Tierra Farris1,2, Paige S. Dunphy2, Bing Zhu2, Clay E. Kibler2 and Jere W. McBride1,2 Departments of Pathology1 and Microbiology and Immunology2, University of Texas Medical Branch, Galveston, Texas

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. Previously, we reported that the E. chaffeensis effector TRP32 interacts with multiple host nucleus-associated proteins including transcriptional regulators, HHEX and DAZAP2, and also auto-activates reporter gene expression in yeast. Here, we investigated TRP32’s function as a nucleomodulin, and demonstrated that it binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C- terminal tri-tyrosine motif. Genistein inhibited TRP32 nuclear entry and Y179 mutants localized primarily to the cytoplasm. Electromobility shift assay demonstrated that TRP32 binds DNA via its tandem repeat domain. TRP32 DNA

75 binding and motif preference were confirmed by supershift assays, and competition and mutant probe analyses. Using ChIP-Seq, we determined that TRP32 binds a specific repeated GG[A/T]GG motif primarily within ±500 bp of gene transcription start sites. Spaced motif analysis confirmed interaction with HHEX and DAZAP2 at the human promoter. Ontology analysis identified enrichment of genes involved in immune cell differentiation, chromatin remodeling, and RNA transcription and processing. TRP32 bound genes (n=1223) are distributed on all chromosomes and include several global regulators of proliferation and inflammation such as FOS and JUN, AKT3 and NRAS, and miRNA 21 and miRNA 142. TRP32 target genes are differentially regulated during infection, with the majority repressed, and direct repression/activation of these genes by TRP32 was confirmed using a luciferase reporter assay. TRP32 is a transcription factor and modulates host expression of genes regulating cell differentiation and inflammation. Manipulating monocyte differentiation may be a mechanism to prevent host cell apoptosis or could serve as a means to avoid immune detection. Future directions include examining the effect of TRP32 on host cell differentiation

Abstract #110 Molecular Detection of Rickettsia Species in Ticks and Fleas in Eastern Missouri and Southern Illinois Catherine Santanello* and Redir Barwari Southern Illinois University Edwardsville School of Pharmacy, Department of Pharmaceutical Sciences

Rocky Mountain spotted fever, the most commonly reported and most severe rickettsial illness in the U.S., is commonly diagnosed in the St. Louis region and in the southeastern U.S. However, it remains unclear if the cases reported are caused by the pathogenic agent, Rickettsia rickettsii, or some other Spotted Fever Group Rickettsiae (SFGR) since diagnostic tests are often cross-reactive with other spotted fever etiological agents. In this study, ticks were collected by flagging through vegetation at local parks and, in addition, ticks and fleas were obtained from domestic pets at local animal shelters. Specimens were sorted by species into pools and were screened by PCR for rickettsial pathogens. Of the 27 pools of Amblyomma americanum representing 270 lone star ticks, 24 pools (88.9%) were positive for Rickettsia amblyommii. Fewer Dermacentor variabilis were collected but of the 7 pools of American dog ticks representing 44 ticks, 100% were infected with Rickettsia amblyommii and 22.7% were infected with Rickettsia montanensis. All pools of cat fleas, Ctenocephalides felis, were infected with Rickettsia felis. These results show that local ticks and fleas are highly infected with R. amblyommii and R. felis, respectively, which might cause rickettsial infection and disease in humans and emphasizes the need for increased awareness and diagnosis.

Abstract #111 Molecular Survey of Arthropod Vectors for Rickettsia in the Eastern Thailand Kamonwan Siriwatthanakul1, Chakrit Hirunpetcharat2, Wuttikorn Rodkvamtook1, Charnchudhi Chanyasanha2, Darunee Utennam1, Pimmada Sakpisal1, Narupon Kuttasingkee1, Yutthapong Sudsawat1, Chanakan Suwanbongkot1 and Jariyanart Gaywee1 1 Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand 2 Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, Thailand

Arthropod-borne diseases are difficult to prevent and control due to variety factors involving disease transmission. Gaining insight of specific arthropod species harboring specific strains of pathogens in particular area will allow public health stakeholders to adopt an effective disease prevention and control. The eastern part of Thailand is an extraordinary region that includes famous tourism beaches and islands, national parks, agriculture fields, industrial parks and the closest border to the capital city. This region also reported high incidence of rickettsioses which is interesting to explore how these diseases were transmitted. A total of 421 pools of blood sucking arthropods were collected from pets, poultry, livestock and rodents in 166 locations of 7 provinces in Eastern Thailand. Six species of ticks (Rhipicephalus sanguineus, R. haemaphysaloides, Boophilus microplus, Dermacentor sp., Haemaphysalis sp., and Ixodes sp.), 3 species of fleas (Ctenocephalides felis orientis, C. felis felis and Echidnophaga gallinacean) and 6 species of lice (Chelopistes meleagridis, Haematopinus eurysternus, Heterodoxus spinigerum, Liperus caponis, Menopon gallinae and Trichodectes canis) were identified. Using real-time PCR, Rickettsial 17 kDa was detected in 44.4% (187/421) of collected arthropods composed of 44/217 tick pools, 128/158 flea pools and 14/46 louse pools. Sequence analysis demonstrated pathogenic Rickettsia marmionii, in R. sanguineus ticks and Rickettsia sp. Cf1, 5 infected R. sanguineus and C. felis orientis. The survey results 76 were subsequently analyzed with geographical information to generate risk area maps, a useful tool for epidemiology warning of arthropod borne diseases in those areas.

Abstract #112 Rickettsia cf sp. Infection in Thailand Jariyanart Gaywee1*, Wuttikon Rodkvamtook1, Walairat Pronwiroon2, Darunee Utainnam1, Piyanate Salyakumthorn1, Pimmada Sakpisal1, Narupon Kuttasingkee1, Kamonwan Siriwatthanakul1, Chanakan Suwanbongkot1, Allen L. Richards3 and Suradet Jaruchinda1 1Armed Forces Research Institute of Medical Science, Bangkok, Thailand; 2Faculty of Science and Technology, Prince of Songklanakarin University, Pattani, Thailand; 3Naval Medical Research Center, Maryland, USA

Rickettsioses are one of the health problems in Thailand. In response to this concern, we have continuously conducted a surveillance program aiming to survey for pathogenic rickettsiae and to investigate of how these bacteria transmitted to human in Thailand since 2004. Using molecular approaches, we have found a varieties species of both Typhus group and Spotted fever group Rickettsia infected human and blood sucking arthropods, ticks, fleas and lice. Interestingly, arthropod survey demonstrated that Rickettsia sp. Cf 1, 5 was the major species detected in fleas (90%) and also found in ticks and lice. Other rickettsial species such as R. typhi and R. felis was found in fleas, R. honei, R. japonica, R. rickettsii, R. marmionii were detected in ticks. These results were concordant with human surveillance information. Besides from pathogenic R. typhi, R. honei, R. japonica, R. rickettsii, R. marmionii and R. felis detected in febrile patients, Rickettsia sp. Cf 1, 5 was recently found in most of tested febrile patients’ blood specimens. Information obtained is essential for health authority to establish an effective prevention and control strategy in Thailand.

Abstract #113 Interaction of Rickettsia felis and Wolbachia endosymbionts in cat fleas, Ctenocephalides felis Krit Jirakanwisal*, Supanee Hirunkanokpun, and Kevin R. Macaluso Vector-borne Disease Laboratories, Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803

Cat fleas (Ctenocephalides felis) have been identified as a biological vector and reservoir of Rickettsia felis. Wolbachia spp. are bacterial endosymbionts that are estimated to infect more than 60% of insect species, including cat fleas. The interaction of vertically transmitted endosymbionts within arthropods may influence the dissemination of pathogenic bacteria. However, the interaction between R. felis and Wolbachia spp. in cat fleas has not been well studied. Thus, we hypothesize that if Wolbachia endosymbionts compete with pathogens for stable vertical transmission events in the vector, then the presence of Wolbachia will influence vertical transmission of R. felis in cat fleas. In order to assess this interaction, fleas were separated and treated with different concentrations of tetracycline to remove Wolbachia from insects. After treatments, fleas were allowed to mate and divided in to three groups. One group was exposed to R. felis with low dose and another with high dose, with a third group serving as a R. felis-uninfected control for 24 hours. Eggs were collected and allowed to develop to adults, and then the newly emerged adults were assessed for vertical transmission of R. felis by quantitative real-time PCR based on the rickettsial 17-kDa antigen gene. Results show that the optimal concentration of tetracycline were 1.0-2.0 mg/ml. Vertical transmission of R. felis and Wolbachia endosymbionts infected fleas was demonstrated and implied a possible influence of Wolbachia on the vertical transmission of this emerging rickettsial pathogen.

Abstract #114 Mechanical infection of Leptotrombidium spp. mites with human pathogenic strains of Orientia tsustsugamushi Piyada Linsuwanon1,*, Elizabeth Wanja1, Surachai Leepitakrat1, Taweesak Monkana1, Piyanate Sunyakumthorn2, Daniel Paris3, Silas Davidson1, Opas Thachin1, Allen Richards4, and James Jones5 1Department of Entomology, Armed Forces Research Institute of Medical Science, Thailand; 2Dep’t of Veterinary Medicine, Armed Forces Research Institute of Medical Science, Thailand; 3Mahidol-Oxford Research Unit, Faculty of

77 Tropical Medicine, Mahidol University, Thailand; 4Viral and Rickettsial Diseases Dep’t, Naval Medical Research Centre, Silver Spring, Maryland; 5Department of Enteric Disease, Armed Forces Research Institute of Medical Science, Thailand

Scrub typhus is an acute mite-borne rickettsiosis that occurs across the Asia-Pacific region. The aim of this study was to investigate if the causative agent, Orientia tsutsugamushi (Otsu), can be injected and mechanically established in Leptotrombidium mite colonies. Laboratory-reared L. chiangraiensis tritonymphs and adult virgin females were injected with approximately 0.07-0.1 µl of Otsu Karp strain culture (1.95x1028 genomic equivalents/ µL) that had been originally isolated from an infected human. Injections were performed into the dorsal or genital regions of tritonymphs and at the dorsal surface of the opisthosomal haemocoel or ventral shield in adults. Mites were not noticeably affected by the injection procedure and there were no effects on oviposition or spermatophore production in adult mites. Real-time PCR of the 47 kDa type-specific antigen encoding gene indicated that mites were consistently infected with Otsu and that there was successful transtadial transmission from nymphs to adults and transovarial transmission from females to eggs and larvae. Inoculated females had persistent Otsu infections for up to 60 days. Larval progenies (15 pools of 150 chiggers from 10 mating pairs) from injected females were infected with Otsu at a rate of 66.7%. Additional experiments including screening a larger samples size of larvae from infected adults and determination of the transmission efficiency of larvae using a mouse model are planned to confirm these findings.

Abstract #115 Investigations into the dynamics of Coxiella burnetii infections and possible vaccination options to control the disease in intensively-reared goats Michael Muleme1,2, Gemma Vincent2, Alexander Cameron1, Joanne Devlin1, Angus Campbell1, Colin Wilks1, Stephen Graves2, Mark Stevenson1, Simon Firestone1, John Stenos2* 1Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Melbourne, Victoria, Australia; 2The Australian Rickettsial Reference Laboratory, Geelong, Victoria, Australia

Coxiella burnetii causes Q fever, a zoonotic disease commonly associated with exposure to infected animals, particularly ruminants. In this study, we elucidated the dynamics of C. burnetii infections by detecting serological responses to C. burnetii in adult and kid goats on a large dairy enterprise in Australia on which 24 human cases of Q fever had occurred since 2013. An indirect immunofluorescence assay (IFA), initially developed for human serology, was adapted and standardised to detect IgG and IgM antibodies to phase 1 and to phase 2 antigens of C. burnetii. We then undertook a cross-sectional study in 164 pregnant goats of different parities (86 primiparous and 78 multiparous) to determine the changes in seroprevalence over the kidding period. Following kidding, the seroprevalence in adult goats increased by 17.7%; the increase was higher (26.7%) in primiparous than in multiparous goats (7.7%) as 47.4% of the multiparous goats were seropositive pre-partum compared to 4.7% of the primiparous goats. However, among adult goats that were seronegative pre-partum, the risk of seroconversion was comparable in multiparous and primiparous goats (24.4% and 26.8%, respectively), suggesting constant risk across the kidding period, irrespective of parity. A longitudinal study in 95 kids from two successive kidding seasons was performed to elucidate the infection dynamics of C. burnetii. Results demonstrated that maternally-derived antibody was detected after feeding on pooled goat colostrum but this subsided within 9 weeks and new infections (shown by a rise in phase 2 IgM) occurred soon after. As kid goats were infected very early in life and well before they were mated, it was concluded that there is a need to vaccinate young goats (before 9 weeks) in addition to the conventional strategy of vaccinating adult goats shortly before breeding. Since a vaccine registered for use in goats is not currently available in Australia, an inactivated autogenous vaccine, prepared from an isolate from the goat farm, was evaluated in 7 to 11 week old goats. Two injections, four weeks apart elicited specific IgM and IgG responses in all vaccinated goats (n=11), while no antibodies were detected in two control groups (n=22). Swelling at the site of inoculation was observed in all of the vaccinated and in 91% of the control group but receded after 3 weeks. The data indicated that this autogenous vaccine could be suitable for immunising young goats (less than 3 months of age) to break the cycle of infection with C. burnetii on this property although further trials to determine level of protection against field challenge are required.

78 Abstract #116 Comparison of lethal and nonlethal mouse models of Orientia tsutsugamushi reveals T-cell population-associated cytokine signatures correlated with disease or protection Alison Luce-Fedrow*1,2,3, Suschsmita Chattopadhyay1,3, John B. Patton4, and Allen L. Richards1,5 1 United States Naval Medical Research Center, Silver Spring, MD 20910; 2 Shippensburg University, Shippensburg, PA; 3 Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD; 4 Thomas Jefferson University, Philadelphia, PA; 5 Uniformed Services University of the Health Sciences, Bethesda, MD

The antigenic diversity of Orientia tsutsugamushi as well as the interstrain difference(s) associated with virulence in mice enhance the necessity to dissect the host immune response. In this study we compared the host response in lethal and non-lethal murine models of O. tsutsugamushi infection using the two strains Karp (New Guinea) and Woods (Australia). The models included the lethal model: Karp IP (intraperitoneal) challenge; and the nonlethal models: Karp ID (intradermal), Woods IP, and Woods ID challenges. We used multicolor flow cytometry to analyze the CD4+ and CD8+ T cell populations and their intracellular production of the cytokines IFNγ, TNF, and IL2 (single, double, and triple combinations). We hypothesized that distinct cytokine signatures could be determined when comparing our lethal model (Karp IP) to our nonlethal models (Karp ID, Woods IP, and Woods ID). Interstrain (Karp IP to Woods IP or Karp IP to Woods ID) and intrastrain (Karp IP to Karp ID) model comparisons were made at distinct time points based upon the severity of illness in the mice: (1) Karp IP, 10 days – onset of severe illness; (2) Karp IP, 14 days – severe illness; (3) Karp ID, 10, 14, 21 days and Woods IP, ID, 10, 21 days – mild to no illness. In our interstrain comparisons of CD4 populations, we observed significant increases in CD4 populations producing IFNγ-IL2 and IFNγ-IL2-TNF in the lethal model beginning at 10 dpi and continuing through 14 dpi. When comparing the nonlethal Woods ID model (21 dpi) to the lethal model at both 10 and 14 dpi, we noted two significant cytokine signatures associated with the nonlethal model - significant increases in the CD4 populations producing IL2 and IL2-TNF. Our intrastrain comparisons of lethal to nonlethal models (Karp IP vs. Karp ID) of CD4 populations revealed similar cytokine signatures as seen in our interstrain comparisons. In particular, significant increases in CD4 populations producing IFNγ-IL2 and IFNγ-IL2-TNF were observed in the lethal group. In addition we observed significant decreases in the overall populations of CD4 cells in the nonlethal group at both 10 and 14 dpi. Interstrain comparison of CD8 populations were dominated by cytokine signatures that included significant increases in overall CD8 populations and in the population of CD8-producing TNF cells in the lethal model at 14 dpi when compared to Woods IP or ID (both nonlethal). In addition, the cytokine signature associated with the Woods ID nonlethal model included an increase in the CD8 populations producing IL2-TNF at 21 dpi, similar to the observations in CD4 populations. The cytokine signatures associated with our lethal model will become depletion targets in future experiments; those signatures associated with our nonlethal model are hypothesized to be related to the protective nature of the nonlethal challenges. Analysis of bacterial loads, Th1 and Th2 cytokines profiles, and seroconversion dynamics are currently underway for correlation to the lethal versus nonlethal comparisons.

Abstract #117 Disease Course and Immune Response of Scrub Typhus in Rhesus Macaques to Increasing Doses by Intradermal Inoculation of Orientia tsutsugamushi Piyanate Sunyakumthorn1*, Manutsanun Sumonwiriya2, Rawiwan Im-erbsin1, Tippawan Anantatat2, Kesara Chumpolkulwong1, Sirima Wongwairot2, Ajchara Vongsawan2, Susanna J. Dunachie2,5, Eric D. Lombardini1, Robin L. Burke1, Allen L. Richards3,4, Nicholas P. J. Day2,5, Daniel H. Paris2,6 1Department of Veterinary Medicine, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; 2Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; 3Dep’t of Viral & Rickettsial Diseases, Naval Medical Research Center, MD, USA; 4Dep’t of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences, MD, USA; 5Centre for Tropical Medicine and Global Health, Nuffield Dep’t of Medicine, Oxford University, Oxford, UK; 6Nuffield Dep’t of Clinical Laboratory Sciences, Oxford University, Oxford, UK

Scrub typhus is an endemic disease in the Asia-Pacific region caused by Orientia tsutsugamushi which can be transmitted to humans by the bite of larval Leptotrombidium mites (chiggers). Broadly protective long lasting vaccines for scrub typhus are not currently available. To further their development, well-characterized animal models and well-understood

79 pathophysiological mechanisms are required. In order to evaluate the efficacy of candidate vaccines in laboratory animal models, it is critical to establish standardized disease parameters and animal study endpoints. In this study, we evaluated the bacterial dissemination dynamics, clinical features and immune responses of rhesus macaques (Macaca mulatta) using three different inoculation dosages of O. tsutsugamushi Karp strain. Four groups of animals (n= 5 per group) were inoculated by intradermal injection in the anterior thigh with different doses of egg-yolk 6 7 7.8 derived infected inoculums; 10 , 10 and 10 muLD50 O. tsutsugamushi Karp or mock inoculum. After inoculation, a series of time course investigations involving bacterial quantitation, antibody response, and immunological bioassays were performed, until day 28 when tissues were collected for pathology and immunohistochemistry. All O. tsutsugamushi-inoculated animals developed classical scrub typhus signs including eschar formation, regional lymphadenopathy, elevation of body temperatures, and bacteremia. The higher dose groups (107 and 107.8) demonstrated more prominent eschars and higher bacteremia levels. The ex-vivo IFN-γ ELISpot assays of peripheral blood mononuclear cells from days 7, 14, and 28 showed significant increase of IFN-γ secreted PBMCs in all O. tsutsugamushi-inoculated animals compared to the control group. High titers (1:3,600) of specific IgM/IgG-antibody against O. tsutsugamushi were observed early on day 14 in the 107 and 107.8 groups while levels in the 106 group peaked at day 21. In addition, on day 7 serum levels of the anti-inflammatory cytokines IFN-γ and IL15 were significantly elevated in the highest dose group (107.8) compared to other groups (p<0.05). The surrogate endpoints provided by these bioassays will be used to develop a disease severity grading system for this animal model, facilitating its use in evaluation of novel scrub typhus drugs and vaccines.

Abstract #118 DNA vaccine candidates conjugated with T cell memory enhancer to study its effectiveness for long term protection Hua-Wei Chen2 , Zhiwen Zhang2, Chye Chan2, Chien-Chung Chao1,2 and Wei-Mei Ching1,2* 1Viral and Rickettsial Diseases Department, Infectious Diseases directorate, Naval Medical Research Center, Silver Spring, MD; 2Uniformed Services University of the Health Sciences, Bethesda, MD

Tetanus toxin (TT) C-terminal fragment (FC) has been shown to enhance various vaccines by eliciting robust immune responses. The conjugation or co-expression of TTFC in DNA vaccine candidates have been shown to lead to the induction of T cell-dependent responses alongside long lasting and stable immunological memory. Previously, we have developed two promising DNA vaccine candidates and in a lethal challenge model. One is pVR1012-r56 which expresses the variable 56 kDa outer membrane protein and the other is pVR1012-47E which expresses a conserved 47 kDa antigen belong to the HtrA heat shock protein family. The 56kDa gene (aa 80 to 461) was fused to the N-terminal of TTFC (451 aa) directly and cloned into VR1012 to produce vaccine candidate (pVR1012-r56-TTFC). The 47E gene (aa 236 to 466) was fused to the N- terminal of TTFC and cloned into VR1012 in a similar way to construct pVR1012-47E-TTFC. Western blot was used to confirm the resulting plasmids did express TTFC fusion proteins in transfected cells. A total of 36 C3HeB/FeJ mice were divided into 6 groups (6 mice each) to evaluate the TTFC’s effectiveness for long term protection. The mice were -TTFC, pVR1012-r56, pVR1012-r56-TTFC, pVR1012-47E, and pVR1012-47E-TTFC) and challenged with 500 x LD50 of live O. tsutsugamushi 12 weeks after immunization. Mice from all six groups developed symptom at day 8 after challenge, no mice survived the challenge after day 13. No enhancement of long term protection was observed from the groups immunized with 47E-TTfc or r56-TTfc as compared to 47E or r56 alone. Abstract #119 Antibiotic efficacy against Coxiella burnetii in axenic media Gilbert J. Kersh and Charles Evavold Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta GA 30333

Coxiella burnetii infection causes the disease Q fever, which can exist in acute and chronic forms. The chronic form of Q fever can persist for years, and the current recommended antibiotic treatment regimens for chronic Q fever require at least 18 months of therapy with doxycycline plus hydroxychloroquine. The hydroxychloroquine is thought to act by increasing the pH of acidic vacuoles where C. burnetii replicates, and allowing better antibiotic function at higher pH. However, the growth, survival, and antibiotic efficacy against C. burnetii under more basic conditions has not been assessed. To evaluate this, C. burnetii was cultured in ACCM-2 media where the pH was varied from 4.50 up to 7.50. Growth of C. burnetii was assessed by real-time PCR at the end of 1-week cultures. Growth of C. burnetii was also measured

80 in ACCM-2 at a variety of pH in the presence of antibiotics. Viability was determined by establishing antibiotic-free secondary cultures after one week of treatment and measuring growth in the secondary cultures by real-time PCR. C. burnetii strain Nine Mile Phase 2 grew well in media up to pH 5.5 but did not grow in media at pH 6.0 and higher. C. burnetii remained viable at the end of the week-long cultures at all pH values. Antibiotic efficacy at different pH was evaluated for strains Nine Mile Phase 2, Nine Mile Phase 1, and Q212 (G strain). Doxycycline and rifampicin showed strong bactericidal activity against C. burnetii independent of pH. Moxifloxacin was bacteriostatic at all pH, and its efficacy did not change as the pH was increased. The data suggest that the effects of these antibiotics on C. burnetii are not enhanced by increasing pH, and that hydroxychloroquine may have effects on C. burnetii survival in vivo that extend beyond altering the pH of vacuoles.

Abstract #120 Development of highly sensitive Quantitative PCR for detection of Bartonella bacilliformis by targeting a multiple- copy DNA sequence Hua-Wei Chen2* , Ai Mochida1, Philip Ching3, Chien-Chung Chao1,2 and Wei-Mei Ching1,2 1Viral and Rickettsial Diseases Department, Infectious Diseases directorate, Naval Medical Research Center, Silver Spring, MD; 2Uniformed Services University of the Health Sciences, Bethesda, MD; 3Aplix Research Inc, N. Potomac, MD

Carrion’s disease is caused by the infection of Bartonella bacilliformis. The sandfly is the transmitting vector. Untreated cases of the acute phase of disease are life-threatening with fatality rates as high as 88%. PCR based diagnostic assays have been developed for detecting B. bacilliformis DNA in clinical samples. Previously, a quantitative PCR (qPCR) targeting pap31 gene, a single-copy DNA sequence, has the analytical detection limit of 10 bacteria. In this study we tried to develop a qPCR targeting a multiple-copy DNA sequence to reach a higher sensitivity. Genome sequence analysis of B. bacilliformis has identified a segment which is present at three different locations. The detection limits were evaluated by three different sets of primers. The best set of primer yielded the detection limit of 3.3 bacteria. DNA extracted from sandflies fed on blood agar containing B. bacilliformis was also tested. Flies fed on Day 1 and 3 were determined as positive for B. bacilliformis; the results were consistent with the earlier study targeting pap31 gene. We investigated the consistency of the newly developed qPCR targeting the multiple-copy DNA segment using samples containing 8.3 or 3.3 copies of genomic DNA. We demonstrated that 18 out of 36 reactions were positive for samples containing 8.3 copies of genome; similarly 12 out of 36 reactions were positive for samples containing 3.3 copies of genome. But only 8 and 2 reactions out of 36 reactions showed positive using primers targeting pap31, respectively. These results showed that quantitative PCR using a multiple-copy DNA sequence is more sensitive for detecting B. bacilliformis in samples.

Abstract #121 Molecular diagnostics of Q-fever at the Laboratory of the Ministry of Agriculture M. Kokhreidze1*, N. Vepkhvadze1, M. Donduashvili1, E. Mamisashvili1, L. Gelashvili1, M.J. Stuckey2, B.B. Chomel2 1 Laboratory of the Ministry of Agriculture, Tbilisi, Georgia 2 Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA

Q-fever is a highly contagious zoonotic disease, caused by Coxiella burnetti a Gram-negative bacterium. This agent is an obligate intracellular pathogen, which belongs to the Coxiellaceae family. Shedding of Coxiella burnetti into the environment occurs mainly during parturition. Urine, feces, and milk may also contain the pathogen. It is a challenge to isolate Coxiella burnetti as it requires Biosafety Level 3 (BSL-3) laboratory facilities. Polymerase Chain Reaction (PCR) targeting the IS1111 insertion sequences is one of the rapid methods for molecular detection of Coxiella burnetti in clinical samples. Primer pairs and probes are used for the RT-PCR assay. (Cox F 5’ GTC TTA AGG TGG GCT GCG TG; Cox R 5’ CCC CGA ATC TCA TTG ATC AGC and TaqMan probe FAM- AGC GAA CCA TTG GTA TCG GAC GTT TAT GG-TAMRA). Samples were collected in all regions of Georgia from both cattle and small ruminants. The samples collected included milk, blood, and genital swab. The molecular biology department performed DNA extractions using Qiagen DNA Blood and Tissue kits on 3,932 blood and 582 swab samples. All material was inactivated at 90°C for 30-60 minutes. Results of RT-PCR tests for 100 samples were negative. Currently, the laboratory is performing work to improve methods of DNA extraction from clinical samples, particularly from milk, which will facilitate more sensitive detection of Coxiella DNA. Detection of DNA of Coxiella

81 burnetti with RT-PCR is a new method for the Laboratory of the Ministry of Agriculture. Implementation of this method will ensure improvement of diagnostics and epidemiological surveillance in Georgia.

Abstract #122 Development and clinical validation of real-time PCR assays for the detection of Ehrlichia species in human specimens IH Chung, AL Austin, CY Kato* Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, GA 30329

Human ehrlichiosis is caused by obligate intracellular gram-negative bacteria, Ehrlichia. Occurring within 1-2 weeks after infection, symptoms are non-specific and can include fever, headache, myalgia, malaise, chills, and in some instances rash. Ehrlichiosis has a low fatality rate, but the risk increases with delayed treatment or when the immunocompromised are infected. Ehrlichiosis is considered an emerging infectious disease due to the increased number of reported cases. Normally causing serious disease in dogs, E. ewingii and E. canis have been reported in humans over the past 2 decades. In 2009, E. muris-like agent was discovered as a human pathogen in the US. The increase in agents of disease, non-specific presentation, possible fatal outcome in untreated patients, and rising number of cases stress the need for early detection. We developed and validated new real-time PCR assays for the detection of Ehrlichia species (PanE1 and PanE2) and E. chaffeensis (ECh2 and ECh4). Targeting different regions of the conserved 16S gene, PanE1 and PanE2 detect Ehrlichia species DNA including E. chaffeensis, E. ewingii, E. muris, E. muris-like, and E. canis. ECh2 and ECh4 are E. chaffeensis specific assays, which target the 16S gene and the disulfide bond (dsb) formation protein gene, respectively. The 16S gene site was previously described for nested PCR by Standaert et al. (2000). In 2005, Doyle et al. used the dsb gene target site for multicolor real-time PCR detection of multiple Ehrlichia species. These regions were used to identify unique TaqMan assay primer and probe binding sites. Primer and probe concentrations were optimized and analytical specificity was tested using exclusivity panels of environmental DNAs (n=35) and bacterial near neighbor DNAs (n=27-31). Known concentrations of E. chaffeensis were spiked into PBS and blood before DNA extraction and tested to evaluate the sensitivity of the assays. The assays have a limit of detection down to 10 copies per reaction. Accuracy was evaluated by testing a blind panel of DNA extracted from blood with high, medium, and low concentrations of Ehrlichia. Results from the blinded panel correlated with expected outcomes. Assay reproducibility was also evaluated, generating consistent results. Clinical verification was assessed by testing 63 previously tested diagnostic specimens with the four assays. Results demonstrated that the new assays are capable of detecting E. chaffeensis, E. ewingii, and E. muris-like agent successfully. The real-time PCR assays are sensitive and specific, serving as effective diagnostic tests for the detection of Ehrlichia in clinical specimens. With the rising incidence of ehrlichiosis, these diagnostic assays will serve as valuable tools for clinical laboratories, especially in areas where the disease is prevalent.

Abstract #123 Rapid diagnostic test for antibody with Korean isolated Anaplasma phagocytophilum from Apodemus agrarius Jun-Gu Kang1, Sung-Suck Oh1,2, Miyoung Yang3, Sungjae Kim3, Jeong-Byoung Chae1, Yong-Sun Jo1, Yun-Kyung Cho, Do- Hyeon Yu4, Jeongmi Kim3, Joon-Seok Chae1* 1Laboratory of Veterinary Internal Medicine, BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National University, 1 Gwanak‐ro, Gwanak‐gu, Seoul 08826, Republic of Korea; 2Incheon Metropolitan City Institute of Health and Environment, 471 Seohyaedae‐ro, Joong‐ gu, Incheon 22320, Republic of Korea; 3VetAll Laboratories, #B‐305 Samsong Techno Valley, 140, Tongil‐ro, Deogyang‐gu, Goyang‐si, Gyeonggi‐do 10594, Republic of Korea; 4College of Veterinary Medicine, Chonnam National University, 77 Yongbong‐ro, Buk‐gu, Gwangju 61186, Republic of Korea

Human granulocytic anaplasmosis, an emerging infectious disease in the Republic of Korea (ROK), is caused by an obligate intracellular tick-borne bacterium of the family Anaplasmataceae. Anaplasma phagocytophilum was found in a variety of animal species including wild deer, cats, dogs, gray squirrels, horses, and mice. The objective of the present study is to develop new/rapid diagnostic techniques with Korean isolate, A. phagocytophilim. A. phagocytophilum MSP2/P44 gene was amplified for production of recombinant protein, excluding hyper variable region. The amplicon was cloned with pGEX-4T-1 vector followed by transformation into E. coli JM109. The purified plasmid DNA was transferred into E. coli BL21. MSP2/P44 was expressed in LB containing 50 mg/ml ampicillin to OD600 = 0.5 followed by induction with 1M IPTG 82 for 12 h at 37℃. Purified recombinant MSP2/P44 was acquired by Ni2+ affinity chromatography. Based on immuno- chromatography and direct sandwich methods, the prototype that can detect the canine antibody against A. phagocytophilum was produced. Using positive sample against A. phagocytophilum, the sensitivity was analyzed by serial dilution method. The positive serum against Fuller and VMRD E. canis strain were used to evaluate the cross-reactivity of the prototype. Western blots of MSP2/P44 in E. coli showed a fusion protein of approximately 59 kDa containing an N- terminal GST tag. The MSP2/P44 protein was purified using Ni2+ affinity chromatography and the purity was confirmed using SDS-PAGE. The 0.39 ug/strip concentration of capture antigen was optimized to achieve high sensitivity and reliability of the test. The optimal amount of anti-dog IgG and mouse IgG gold conjugate pad was 0.357 ug/strip. Basing the data, the prototype has been produced. The visual detection limits of the prototype were defined as the minimum concentrations causing the color density of the test lines distinguishably weaker than those in the assay of the negative control sample. 1:800 ratio of positive sample caused a slight but distinguishable difference compared to the negative control. Although the strip assay sensitivity was lower than the microplate-based ELISA format, the strip assay is fast, simple, and portable, providing a much needed tool for on-site monitoring of A. phagocytophilum in dogs. The cross reactivity of prototype was not found in E. canis strains. A one-step strip immunoassay has been developed and can be utilized in rapid detection of A. phagocytophilum in dogs. With respect to its overall speed and simplicity, it is more economical to perform on large numbers of samples, this prototype can be used for diagnosis of A. phagocytophilum infection in Veterinary Medicine.

Abstract #124 Molecular and serological evidence of Anaplasmosis in South Korea Seung Hun Lee, Hae kyung Lee, Yeong Seon Lee, Seon do Hwang* Division of Zoonoses, Center for Immunology and Pathology, Korea National Institute of Health, Korea Centers for Disease Control and Prevention, Cheongju-si, Chungcheongbuk-do 28159, Republic of Korea

Human Granulocytic Anaplasmosis (HGA) is a zoonotic tick-borne disease and the agent is known as Anaplasma phagocytophilum which is mainly transmitted by ticks. In South Korea, there are growing concern about the possible emergence of HGA since reporting A. phagocytophilum patient in 2014. This report shows the recent laboratory findings of HGA in South Korea. Patients samples for suspected with anplasmosis were examined using IFA or PCR for A. phagocytophilum. Serological test of A. phagocytophilum was performed by IFA using commercial kit. For molecular detection of A. phagocytophilum infection, PCR was performed using 16S rRNA, ankA, msp2/p44 genes and those were further analyzed through cloning and phylogenetic analysis. Morulae were observed in peripheral blood smear using Diff- Quik staining. In serological tests of acute-phase serum using IFA, 14 of the 118 cases were detected IgG or IgM for A. phagocytohilum and 3 of 6 cases were showed seroconversion. Three of the 118 cases showed positive result of PCR test and could find morulae in 2 of 3 cases blood samples and we isolated 2 cases using these A. phagocytohilum positive samples. In phylogenetic analysis these cases of Korea are generally associated with other Asian stains. This study is meaningful that we could evaluate the status of A. phagocytohilum and it provides information of molecular basis A. phagocytohilum in South Korea.

Abstract #125 Clinical characteristics of rickettsiosis in southern Mexico César Lugo-Caballero1*, Karla R. Dzul-Rosado1, Adolfo Palma-Chan2 and Jorge Zavala-Castro1 1Regional Research Center “Dr. Hideyo Noguchi,” Autonomous University of Yucatán, Mexico 2Regional Hospital Agustín O’Horán, Mérida Yucatan Mexico

There are an increasing number of patients with fatal or severe rickettsiosis that have been reported in southern and northern regions of Mexico. In Yucatan, the ecologic characteristics of vectors related to domiciliary transmission suggest than R. felis or R. tiphy should be the most prevalent etiological agents however, as it has been reported by our group, the clinic manifestations could be different from those observed elsewhere. Therefore, the objective of this work was to determine the clinic and laboratory characteristics of patients that were diagnosed from 2013 to 2015. Fever and a petechial exanthema were the first manifestations followed by joint pain. 10% of the patients also showed neurologic 83 manifestations like dyslalia and aphasia that disappeared after treatment with doxycycline. Laboratory findings were inespecific. The age group between 5 to 12 years old was the more affected by this disease. Almost all of the cases resulted from R. tiphy infection including the severe cases and surprisingly, only a few were caused by R. rickettsii. This suggests the urge of a deeper comprehension of the ecologic cycle of these particular species in our region.

Abstract #126 Social intervention against tick-borne diseases in a rural community of Mexico Karla R. Dzul-Rosado1*, César Lugo-Caballero1, Juan Jose Arias-Leon2, Gaspar Peniche-Lara2 and Jorge Zavala-Castro1 1Regional Research Center “Dr. Hideyo Noguchi,” Autonomous University of Yucatán, Mexico 2 Faculty of Medicine, Autonomous University of Yucatan, Mexico

Ticks are the second most important vectors not only because its worldwide distribution but also for the severe diseases that can transmit like rickettsiosis among others. People living in neglected communities, have a closer contact with ticks due to its economical activities like the agriculture, cattle-trade, hunting; or due to its poor living conditions. Therefore, intervention campaigns against vector borne diseases should consider the people knowledge and habits in the strategies aimed to reduce their exposition to those vectors. The objective of this work was to develop a pilot strategy that could be useful for the social intervention of Mayan communities from Yucatan, which considering their social and ecological characteristics are under constant risk of acquire tick borne diseases. This work has been developed in two phases. During the first phase we collected and analyzed the social determinants to design intervention campaigns against tick-borne diseases. The second phase consisted in the social intervention of children through talks, social cartography and ludic activities; and adults by workshops and community participation collects. Both interventions led to a strong empowerment of the knowledge by these people, which could have an impact in the reduction of tick-borne diseases.

Abstract #127 Generation of humanized chimeric IgG and IgM antibodies specifically recognize the 56 kDa protein antigen from Orientia tsutsugamushi Chien-Chung Chao1,2, Zhiwen Zhang1,2, Tatyana Belinskaya1,2, and Wei-Mei Ching1,2 1Viral and Rickettsial Diseases Department, Infectious Diseases Department, Naval Medical Research Center 2Department of Preventive Medicine and Biostatistics, Uniformed Services University of the Health Sciences

Scrub typhus disease is an acute febrile illness that is caused by the gram negative intracellular bacteria, Orientia tsutsugamushi. Clinical manifestations of scrub typhus vary widely from a mild and self-limiting febrile illness to a more severe illness that may be fatal. Traditionally, the diagnosis of scrub typhus mainly relies on serologic tests. The disease could be diagnosed retrospectively using IFA as the gold standard in cases of seroconversion or a >4-fold rise in antibody titers between acute-phase and convalescent-phase serum specimens. Scrub typhus are under reported due to the nonspecific symptoms and lack of an easy to perform diagnostic tests. Recent effort between NMRC and InBios has lead to the development of Scrub Typhus DetectTM IgG or IgM Rapid Test (ST DetectTM IgG /IgM ) using 3 patented recombinant proteins to detect Orientia specific IgG or IgM antibodies. An attempt was made to generate humanized chimeric antibodies that could be used as positive controls for ST DetectTM IgG /IgM Rapid Test. The recombinant 56 kDa protein antigen derived from Karp strain of Orientia was used to immunize mice and the monoclonal antibody producing clones were screened for specific and high avidity IgG and IgM. The selcted IgG and IgM producing clones were then used to genearte the hmanized chimeric antibodies through series processes which include sequencing, cloning, expression, and purification. The purified chimeric IgG and IgM have been evaluated for their reactivity with r56 antigen in ELISA and ST DetectTM IgG /IgM rapid test. The results showed that the titer for IgG was greater than 10,000 while that for IgM was around 40 based on ELISA assay using a combination of 3 patented antigens. Addtionally, the the chimeric IgG and IgM were also detected by ST DetectTM IgG /IgM rapid test. The results demonstrated that these chimeric antibodies can be used as positive controls for rapid tests.

84 Abstract #128 Importance of Geographic Relevance in Determining the Diagnostic Cut-off for Scrub Typhus Serology Jamie L. Perniciaro*, Alfred T. Harding, William L. Nicholson Centers for Disease Control and Prevention, 1600 Clifton Rd. Atlanta, GA 30333

Over a million human cases of scrub typhus may occur annually throughout Asia. Diagnosis of scrub typhus is complicated by the use of non-validated assays. Choosing the diagnostic cut-off can influence the number of false positives and false negatives. This study evaluates geographic variation when determining the diagnostic cut-off for the InBios Scrub Typhus Detect IgG enzyme linked immunosorbent assay (ELISA). Using the InBios ELISA, human sera samples (n=360) were tested to detect IgG antibodies reactive with a mix of recombinant p56-kDa protein from four serotypes of Orientia tsutsugamushi. Test samples were originally collected as part of a febrile illness study in Bangladesh, where scrub typhus is considered endemic. Using the cut-off provided by the kit, determined with a limited number of samples from India, 30% of test samples contained reactive antibodies. To establish an ELISA cut-off, the manufacturer recommends calculating the average absorbance plus three times the standard deviation of geographically relevant normal human sera or human sera with unrelated infections. A cut-off was determined using febrile human sera samples from Bangladesh, which previously tested negative (<1/32 titer) by the indirect immunofluorescence assay (IFA) for Rickettsia typhi, Rickettsia conorii, and Orientia tsutsugamushi Karp serotype. An alternate ELISA cut-off was determined using febrile human sera samples collected in the United States. Using the Bangladesh cut-off, 24% of test samples contained reactive antibodies. Using the United States cut-off, 28% of test samples contained reactive antibodies. A subset of Bangladesh sera samples were tested by IFA to detect IgG antibodies reactive with the Karp and Kato serotypes of Orientia tsutsugamushi, which are two serotypes included in the ELISA kit. Ongoing work will observe the repeatability and variation of ELISA results and the percent agreement of IFA and ELISA results.

Abstract #129 Enrichment of Rickettsia for Detection and Increased Sensitivity of Diagnostic Samples Marah Condit and Cecilia Kato* Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta Georgia

Infections by spotted fever group (SFG) Rickettsia species cause serious human illnesses worldwide. Confirmation of these diseases from clinical diagnostic samples can be difficult due to low bacteremia in the acute stage of illness. Circulating R. rickettsii has been previously described to have low copy in peripheral blood of ill patients in values less than 100 bacteria per milliliter of whole blood. Detection at such levels is difficult because of the low number of copies represented in the aliquot tested. The PanR8 real-time PCR assay has a limit of detection (LOD) of ~9 copies per reaction, and although this method is highly sensitive and reproducible, this LOD will only result in a positive with samples containing approximately 1,800 copies per ml of blood; well above previously described concentrations. This leads to false negatives for cases with strong clinical descriptions of SFG rickettsia. In this study Rickettsia conorii (Rco) was used to model a method for detecting low copy number samples by amplification in cell culture. Forty copies of viable Rco was spiked in 6 mL of prepared media and then inoculated into a confluent 25 cm2 tissue flasks of uninfected Vero E6 cells. The supernatant from days 5-8 was sampled in duplicate, followed by DNA extraction and quantitation using the PanR8 assay. Cells were harvested prior to or at the time of the appearance of CPE (<5%) in the monolayer. On the final day, the monolayer was rinsed, lifted, concentrated and extracted to determine copy number of cell associated Rco. The total copy number of Rickettsia in the supernatant was 15.54 ± 11.09 times greater than that which is cell associated on the day of cell harvest. This was repeated with 4,000 and 40,000 copy spikes with similar results of 6.6 and 24 times greater respectively. Rco was detected in the supernatant consistently at Day 4 after inoculation with 40 copies and at Day 1 with inoculation of 4,000 and 40,000 copies. Rco can be consistently detected in the supernatant of cultured isolates within 4 days of inoculation, well before the appearance of CPE in the monolayer. These results indicate that it may be possible to increase the accuracy of confirmatory diagnostic testing for SFG rickettsia agents by introducing a cultivation amplification step, to increase sensitivity for detection in samples with low copy numbers which might otherwise escape detection.

85 Abstract #130 Experimental evolution of Coxiella burnetii using chemostats James M. Battisti, Margie Kinnersley and Michael F. Minnick Division of Biological Sciences, University of Montana, Missoula, Montana

Background-Coxiella burnetii is an extremely infectious, intracellular bacterium that causes Q fever in humans, typically following exposure to the spore-like morphotype. This small cell variant (SCV), is highly resistant to environmental stressors (heat, desiccation, UV light) and does not replicate. Once inside acidified phagolysosome-like compartments of host cells, such as alveolar macrophages, SCVs transition to metabolically-active morphotypes termed large cell variants (LCVs) and initiate cell division. The external cues that drive the SCV-LCV-SCV developmental cycle are largely unknown but are clearly important to understand, as interruption or delay of morphogenesis could significantly impact C. burnetti transmission and virulence. The overall goals of this project are to develop a novel, continuous culture system for C. burnetti, to use this protocol to better understand the exogenous cues driving the SCV-LCV-SCV developmental cycle, and to explore C. burnetii’s molecular response to these environmental factors. Methodology/Principal Findings- To date, we have constructed several continuous culture apparati (chemostats) for axenic culture of Phase II C. burnetii in ACCM-2 liquid medium. Preliminary results indicate a diminished C. burnetti lag phase in the chemostat combined with low-density growth over a 7-day period as compared to standard batch culture. Conclusions/Significance- We hypothesize that because continuous culture systems enable precise manipulation of various aspects of the microbial environment (specifically growth rate, pH, percent O2, temperature and carbon source), we will be able relate exogenous cues to changes in C. burnetii’s physiology and elucidate the molecular signals that govern the SCV-LCV-SCV developmental cycle.

Abstract #131 Anaplasma phagocytophilum 16S rDNA sequences present in domestic dogs from the Mnisi community area, Mpumalanga Province, South Africa Mamohale E. Chaisi1, Samantha K. Wills1, Agatha O. Kolo*1, Kelly A. Brayton1,2, Marinda C. Oosthuizen1 1Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa; 2Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, USA

Anaplasma phagocytophilum is a tick-transmitted, intracellular, Gram-negative bacterium of the family Anaplasmataceae, transmitted by ixodid ticks. It is the agent of tick-borne fever in ruminants, and granulocytic anaplasmosis in a wide variety of domestic animals. The disease recently gained attention in veterinary and human medicine as a re-emerging zoonosis. The enzootic cycle includes rodents, wild ungulates, and possibly birds as reservoir hosts. Humans are accidental hosts; the severity of human granulocytic anaplasmosis (HGA) ranges from mild to severe infections and occasionally death. Dogs are also considered as accidental hosts; infection causes canine granulocytic anaplasmosis (CGA), an acute febrile illness with lethargy and inappetence. The Mnisi community is situated in the north-eastern corner of the Bushbuckridge Municipal Area, Mpumalanga Province, South Africa. Mnisi is a rural, high poverty area located at the livestock/wildlife/human interface of the western boundary of the Kruger National Park. A recent study in this area indicated that dogs may play an important role in life-cycles of zoonotic rickettsial infections. We screened blood samples from 56 domestic dogs originating from the Mnisi community area for the presence of tick-borne haemoparasites using the reverse line blot (RLB) hybridization assay; and for specific detection of A. phagocytophilum using a quantitative real- time PCR (qPCR) assay based on the msp2 gene. The RLB results revealed the presence of A. phagocytophilum (1.79%); Ehrlichia canis (33.93%); E. ruminantium (8.93%); Babesia rossi (12.5%); B. vogeli (5.36%); B. microti (5.36%) and Anaplasma sp. (Omatjienne) (1.79%), either as single or mixed infections. Fifteen (26.79%) samples hybridized with the Ehrlichia/Anaplasma genus specific probe only, and not with any of the species specific probes. Haemoparasite DNA was not detected by RLB in 25% of the samples. The qPCR results indicated the presence of A. phagocytophilum in 82.93% of the samples. In order to determine the relationship between the 16S rRNA sequences of A. phagocytophilum of dogs from this area with published sequences, the 16S rDNA from 15 samples was amplified, cloned and the recombinants were sequenced. Preliminary sequencing results indicate the presence of 16S rDNA sequences closely related to A. phagocytophilum, A. platys, and an undescribed Anaplasma sp. of dogs from South Africa. We furthermore plan to clone and sequence the msp2 gene of A. phagocytophilum to determine whether any sequence variation exist in the target region of the qPCR assay.

86 Abstract #132 High-content imaging reveals increased cellular endocytosis during Coxiella burnetii parasitophorous vacuole maturation Charles L. Larson*, Diane C. Cockrell, and Robert A. Heinzen Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT

Coxiella burnetii is an intracellular bacterium and the causative agent of human Q fever. Pathogen replication within a specialized parasitophorous vacuole (PV) resembling a host phagolysosome is required for pathogenesis. Fundamental to understanding mechanisms of C. burnetii infection is characterization of host membrane trafficking pathways that contribute to PV biogenesis. Several studies have shown dysregulation of endocytic, autophagic, or secretory systems disrupts PV formation. Once C. burnetii is internalized, effector proteins translocated into the host cell via the C. burnetii type 4B secretion system (T4BSS) modify host membrane transport to promote maturation of the nascent PV into a growth-permissive vacuole. Previously, the T4BSS effector CvpA was found to promote C. burnetii growth and PV formation. When ectopically expressed in host cells, CvpA targets clathrin-coated vesicles and inhibits internalization of transferrin (Tf), a well-characterized substrate of clathrin-mediated endocytosis that traffics within the endosomal system prior to being recycled out of the cell. The goal of this study was to understand if modulation of endolysosomal flux occurs during native infection conditions. HeLa cells, infected with C. burnetii or left uninfected, were incubated with fluorescent Tf for 0 to 30 minutes and the amount of Tf internalized by cells was quantitated by high-content imaging. At days three and five, but not at day one, post infection, the mean amounts of Tf internalized by infected cells were significantly greater than uninfected cells. Despite infected cells internalizing more Tf, the time required to recycle Tf out of the cell was the same between infected and uninfected cells. These data indicate C. burnetii PV maturation coincides with increased endocytic flux. Furthermore, this study demonstrates the power of high-content imaging for measurement of cellular responses to pathogen infection.

Abstract #133 Ehrlichia chaffeensis Effector TRP47 Binds Host DNA and is SUMOylated. Clayton E. Kibler1*, Sarah L. Milligan1, Tierra R. Farris1,2, Bing Zhu1, and Jere W. McBride1,2 Departments of Pathology1 and Microbiology and Immunology2, University of Texas Medical Branch, Galveston, TX

Ehrlichia chaffeensis is an obligately intracellular bacterium and is the etiologic agent of human monocytic ehrlichiosis, a life-threatening NIAID emerging tick-borne disease. E. chaffeensis selectively infects and survives within mononuclear phagocytes through undefined mechanisms involving effector proteins that are secreted using a type 1 secretion system and interact with numerous host proteins. One of these effectors, TRP120, is a nucleomodulin that binds host DNA via its tandem repeat domain and alters host gene transcription. Recently, we demonstrated that TRP120 is conjugated with SUMO2/3 isoforms in a C-terminal canonical SUMO motif, a post-translational modification that mediates interactions with host proteins and subcellular localization. TRP47, another effector, is the most highly expressed ehrlichial protein during infection of mammalian cells, was found to interact with multiple functionally relevant eukaryotic proteins, and had been observed in the nucleus. However, it is unclear if TRP47 DNA-binding activity or host-mediated post-translational modification of TRP47 play a role in E. chaffeensis infection. In this study, we investigated the potential nucleomodulin function and SUMOylation of TRP47. Using ectopic expression of various GFP-tagged TRP47 constructs and immunofluorescence assays, we found that TRP47 localizes to the host cell nucleus and a MYND-binding motif in the TRP47 N-terminal region is responsible for nuclear localization in HeLa cells during infection. DNA-binding activity was observed with electrophoretic mobility shift assay (EMSA). In addition, we demonstrated that TRP47 is SUMOylated at lysine 71 by in vitro SUMOylation assay and high-density microfluidic peptide array. These data suggest that TRP47 may be a novel nucleomodulin with functions similar to TRP120. We are currently working to identify TRP47 target genes and the host cell processes they are involved in with high-throughput deep sequencing (ChIP-Seq) and ontology analysis. Future directions include assessing direct modulation of host gene transcription by TRP47 with a luciferase reporter assay, and investigating the role of TRP47 SUMOylation in transcription modulation.

87 Abstract #134 Structure and distribution of VirB6-4 in A. phagocytophilum Francy L. Crosby*, David R. Allred, Yu-Ping Xiao, Donna S. Williams, Anna M. Lundgren and Anthony F. Barbet. Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL

Several pathogenic gram-negative bacteria translocate virulence factors into their eukaryotic hosts by means of a Type 4 Secretion System (T4SS) that spans the inner and outer membranes. Genes encoding components of a type 4 secretion system have been identified in several species of pathogenic bacteria of the order Rickettsiales due to their sequence similarities to prototypical systems such as the VirB system of Agrobacterium tumefaciens. The complete structure of these components at the genomic level was determined in several A. phagocytophilum strains, showing overall conservation between different strains, with the exception of VirB2 and VirB6. The virB6 loci are characterized by the presence of 4 copies arranged in tandem within a single operon (virB6-1 through virB6-4). Interestingly, VirB6-4 varies between different strains due to the presence of extensive repeats found at the C-terminus of this protein. RT-PCR analysis detected transcripts from all four virB6 genes in both infected mammalian HL-60 and tick ISE6 cells. Specific antisera against VirB6-4 were generated from rabbits immunized with a synthetic peptide from the virB6-4 hydrophilic repetitive region. The antiserum reacted in ELISA with the peptide used as the immunogen and immunoblots showed reactivity of the anti-VirB6-4 sera against both infected HL-60 and ISE6 cells. The size of the detected band was consistent with the predicted molecular weight of 470KDa, known from the sequence of the encoding gene. Preliminary proteomics data of co-immunoprecipitated proteins using anti-VirB6-4 sera detected peptides of VirB6-4, two predicted DNA binding proteins APH_0784 and APH_1100 and the outer membrane protein A (OmpA) APH_0338. Surface-specific immunoprecipitation and immunofluorescence microscopy experiments suggest restricted, focal distribution of VirB6-4, with accessibility primarily on the outer surfaces of morulae, consistent with the polar distribution found in A. tumefaciens. Characterization of T4SS components such as VirB6-4 is of great importance to gain a better understanding of the process of translocation of A. phagocytophilum effectors into host cells and to identify effector proteins important for colonization.

Abstract #135 Ehrlichia chaffeensis TRP120 influences Notch signaling and cell cycle by modulating The SCF ubiquitin ligase FBW7 to promote infection Jennifer Y. Wang1, 2, Tian Luo2, Paige Dunphy, Jere W. McBride1, 2 Departments of Cell Biology1 and Pathology2, University of Texas Medical Branch

Human monocytotropic ehrlichiosis (HME) is an emerging tick-born zoonosis caused by the obligately intracellular, gram- negative bacterium, Ehrlichia chaffeensis. E. chaffeensis survival is dependent on secreted tandem-repeat effector proteins (TRPs), which function to manipulate host cellular processes such as cell proliferation and differentiation. Using yeast two-hybrid assay, we previously determined that TRP120 interacts with FBW7 (F-box and WD domain repeat- containing 7), the substrate recognition subunit of the E3 ligase complex SCF (Skp1, Cul1 and F-box). FBW7 is a well- studied protein and functions as a tumor suppressor, targeting several oncoproteins that are involved in cell proliferation and differentiation (such as Notch, cyclin E, JUN and MYC). The purpose of this study was to determine if E. chaffeensis modulates Notch signaling and the host cell cycle through TRP120-mediated degradation of FBW7. We confirmed the interaction of TRP120 with FBW7 by using co-immunoprecipitation, and demonstrated that siRNA knock-down of Fbxw7, resulted in an increased E. chaffeensis load. FBW7 levels were monitored during infection and were consistently decreased compared to control cells. Although a reduction in FBW7 in infected cells was detected, the mRNA levels of FBW7 increased. In addition, increases in Notch1, cyclin E and c-MYC, which are targets of SCF complex, results that suggest a reduction of SCF ligase activity/function. The increase in Notch1 and cyclin E correlated with increased Notch signaling activity and cell proliferation, leading to prolonged host cell survival. These studies support the conclusion that FBW7 stability is modulated by E. chaffeensis TRP120, thereby suppressing innate host defenses and to promote host cell survival. These results also suggest the loss of FBW7 is important in promoting E. chaffeensis survival in the host cells. However, the mechanism of FBW7 degradation during infection is currently unknown. Future studies will investigate the hypothesis that direct interaction between TRP120 and FBW7 lead to the proteasomal degradation of FBW7.

88 Abstract #136 Protein aggregation in Ehrlichia chaffeensis during macrophage infection Dorota Kuczynska-Wisnik1,2, Michal Zolkiewski1, and Roman R. Ganta2* 1Department of Biochemistry and Molecular Biophysics, Kansas State University; 2Center of Excellence for Vector-Borne- Diseases (CEVBD), Department of Diagnostic Medicine/Pathobiology, Kansas State University

Ehrlichia chaffeensis is an obligatory intracellular pathogen transmitted through infected tick Amblyomma americanum to humans and several other mammals. Human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis occurs mainly in the U.S.A, but also has been reported in Europe, Asia, Africa, and South America. The severity of the disease varies from asymptomatic seroconversion to death. To establish infections in mammalian and ticks cells, E. chaffeensis needs the capability to evade the host defenses, but it is still not clear how the pathogen responds to and overcomes the host- induced stress. We have previously shown that the expression of the molecular chaperone clpB in E. chaffeensis is strongly upregulated immediately after infection of mammalian cells. ClpB is an ATP-dependent heat-shock protein with a unique disaggregase activity that reactivates aggregated proteins under cellular stress conditions. Induction of clpB in E. chaffeensis during its replication in mammalian cells suggests that the ClpB activity supports the pathogen survival inside the host, but no link between protein aggregation and a pathogen life cycle has been established so far. We investigated the extent of protein aggregation in E. chaffeensis during infection of canine macrophage cell line, DH82. We discovered that the mammalian cell environment induces misfolding and aggregation of a significant number of Ehrlichia chaffeensis proteins. The size of the aggregated fraction of E. chaffeensis proteins increases during the first 48h post infection and subsequently levels-off. The 48-h post infection window corresponds to the upregulated production of the clpB mRNA. We also found that ClpB and its chaperone partner DnaK accumulate with protein aggregates formed in E. chaffeensis cells during the infectious stage. To disrupt the activity of ClpB in E. chaffeensis, we incubated the infected cells with up to 0.5 mM guanidinium chloride (GuHCl), which inhibits the ClpB ATPase activity in vitro. 0.5 mM GuHCl had no impact on the host cells, whereas the viability of the pathogen was reduced by ~80% in the presence of the inhibitor. Importantly, we found that the size of the aggregated protein fraction in E. chaffeensis increased significantly in cultures supplemented with 0.25 mM and 0.5 mM GuHCl. Thus, we discovered that an exposure of E. chaffensis to the stressful host environment during infection results in aggregation of the pathogen’s proteins. Moreover, we showed that the molecular chaperone ClpB supports the survival of E. chaffensis during the infections stage by reducing the amount of protein aggregates. Altogether, our studies establish a link between the protein quality control and the pathogen survival. (This work was supported by the PHS grant # AI070908 from the National Institute of Allergy and Infectious Diseases, USA. This work is also supported with funds from the NIH/K-INBRE Bridging Grant, University of Kansas and from the CEVBD, Kansas State University.)

Abstract #137 Ehrlichia chaffeensis Mediated Epigenetic Reprogramming of the Host Facilitates Intracellular Survival Shubhajit Mitra*, Paige S. Dunphy, Jere W. McBride Department of Pathology, University of Texas Medical Branch, Galveston, TX

Ehrlichia chaffeensis is an obligatory intracellular bacterium that primarily infects mononuclear phagocytes and causes human monocytotropic ehrlichiosis (HME). E. chaffeensis T1S effectors like ANK200, TRP32, TRP47 and TRP120 interact with diverse array of host proteins to establish a replicative niche inside the host. Recent studies have shown that the TRP120 interacts with host epigenetic machinery involved in post translational modification (PTM) of histones including polycomb repressive complex (PRC). Polycomb repressive complex is a major repressor of Hox genes, involved in cellular proliferation and differentiation. PRC falls into two distinct protein complexes, PRC1 and PRC2 involved in ubiquitination of H2A at lysine 119 (H2AK119ub) and trimethylation of H3 at lysine 27(H3K27me3), respectively. In this study, we have investigated the role of TRP120 in reprogramming of host genes via PTM of histones. Using immunofluorescence microscopy and immunoblotting we have determined that TRP120 recruits Polycomb group RING finger isoforms 1-6 (PCGFs) from nucleus to the ehrlichial vacuole. Comparative analysis of the protein and transcript level of the PCGFs suggest degradation of these isoforms in the cytoplasm of the infected cells with an exception of PCGF2. The temporal dislocation of PCGFs from nucleus to the morulae also coincides with a decrease in the levels of the PRC1-mediated H2AK119ub and differential expression of the Hox genes. An increase in PRC2 mediated H3K27me3 repressive marks was

89 observed during infection, but ChIP showed a gene specific decrease in this mark. The decrease in H3K27me3 can be attributed to another TRP120 interacting partner JMJD3/KDM6B, an H3K27me3 specific demethylase. siRNA knockdown of PCGFs resulted in significant increase in the bacterial load, which demonstrates the significance of PCGFs in ehrlichial survival. However, the complete mechanism involving effector-PcG-mediated epigenetic reprogramming of host is not clear and is currently under investigation.

Abstract #138 Brain Endothelial Activation and CXCR3-Related Gene Expression in Murine Models of Scrub Typhus Thomas R. Shelite*,1, Nicole L. Mendell*,2, Kathryn C.Smith3, David H. Walker2, Donald H. Bouyer2, Lynn Soong1 1Dep’t of Microbiology and Immunology, University of Texas Medical Branch; 2Dep’t of Pathology, University of Texas Medical Branch; 3Ball High School and Center in Environmental Toxicology, University of Texas Medical Branch

Severe scrub typhus is associated with the development of various neuropathies, including temporary or permanent blindness, cranial nerve palsies, deafness, headaches, as well as confusion, delirium, and seizures. Scrub typhus is a neglected disease with approximately one-third of the world’s population at risk of being infected with the etiologic agent, Orientia tsutsugamushi. The bacteria can infect endothelial cells, macrophages, cardiac myocytes, and dendritic cells; however, very little is known regarding immunological and vascular changes in the brain during Orientia infection. In this study, we utilized our newly developed endotheliotropic murine model of scrub typhus to assess the pathological changes and gene expression in the brains of mice with lethal vs. sublethal infection. Female C57BL/6 mice were intravenously (i.v.) challenged with a lethal or sub-lethal dose of O. tsutsugamushi Karp strain (OtK) and monitored for disease severity. While both groups of infected mice had significantly increased IFN-γ, CXCL9, and CXCR3 gene expression than mock controls, there was no significant differences in IFN-γ and CXCL9 levels between lethal and sub-lethal groups. Lethal infection, however, resulted in a significant increase in CXCR3 gene expression, implying accelerated activation of CXCR3-mediated responses in lethally-infected mouse brain. While sub-lethally- infected mice had evidence of endothelial activation in the brain, as assessed by mRNA levels of angiopoietin 2/ angiopoietin 1 ratios, the Ang2/Ang1 ratios in lethal infection was significantly higher compared to sub-lethal infection. In addition, OtK- infected mice had meningoencephalitis, microvascular cuffing, hemorrhage, occlusion, and development of scrub typhus nodules that varied dependent on severity of disease, as reported for human patients. Currently, we are examining whether increased endothelial activation and/or recruitment of CXCR3-expressing T cells to the brain contribute to disease lethality in acute stage and in persistent infection. This study is the first to examine Orientia-induced brain pathology and cytokine/chemokine gene expression in mouse models of scrub typhus and will provide new insights on disease pathogensis.

Abstract #158 Spotted Fever Group Rickettsiae Hijack Annexin A2-mediated Host Fibrinolytic Machinery for their Adherence to Vascular Endothelial Surfaces Tuha Ha1, Nicole Mendell1, Yuan Qiu1, Thomas Shelite2, Guang Xu1, Qinyu Gong1, Thomas Ksiazek1, Jia Zhou3, Vsevolod Popov1, Donald H. Bouyer1,*, Bin Gong1,* 1Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, 2Department of Microbiology and Immunology, 3Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas

Background: Endothelial cells (ECs) are the primary mammalian host target cells of spotted fever group (SFG) rickettsiae. The most prominent pathophysiological effect of infection is increased microvascular permeability, causing vasogenic cerebral edema and non-cardiogenic pulmonary edema with potentially fatal outcomes. The underlying mechanism(s) of rickettsial attachment to, and anchoring on, the EC surface remains incompletely determined, including how the bacteria overcome shear stress from blood flow prior to host cell invasion. Method: In the present study, in an established in vitro rickettsial infection model, we examined the role of the annexin A2-S100A10 complex-based endothelial fibrinolytic machinery during rickettsial adherence to human endothelial cells. Results: We have demonstrated that rickettsiae colocalize with annexin A2 and S100A10 on EC surfaces at the beginning of infection and host plasminogen can bind to rickettsae. We also pave provide direct evidence that the annexin A2-S100A10 complex plays a critical role in the early stages of rickettsial attachment to nonphagocytic host cell surfaces. Conclusion: The in vitro data suggest that the annexin A2-S100A10 complex contributed to rickettsial attachment to ECs. This study lays the groundwork for developing novel host-targeted therapeutics for rickettsiosis, one of the most devastating human infections if left untreated.

90

th 28 Meeting of the

American Society of Rickettsiology

Tuesday June 14, 2016

Abstracts #139-157

Host-Vector Pathogen Interactions ll

Innate and Adaptive Immunity

Genetics, “Omics”, New Technologies, and Tools

Funding for this conference was made possible [in part] by R13 AI126727-01 from the National Institute of Allergy and Infectious Diseases. The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the U.S. Department of Health and Human Services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government.

91 Abstract #139 An Overview of the Molecular Details Governing SFG Rickettsial Pathogenesis Juan J. Martinez Vector-Borne Disease Laboratories, Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 70803

The pathogenesis of spotted fever group (SFG) rickettsiae, upon transmission into the host, depends on the pathogen’s ability to bind to and invade target host cells and ultimately disseminate to target organs and tissues. Analysis of several completed rickettsial genomes has revealed the presence of at least 17 open reading frames (orfs) termed sca (surface cell antigens) whose products exhibit high homology to auto-transporter protein families in Gram-negative bacteria. Five members of this family, Sca0/OmpA, Sca1, Sca2, Sca4 and Sca5/OmpB are highly conserved among most pathogenic SFG rickettsiae and several of them have been shown to be important in the adherence to and invasion of mammalian cells in vitro. Although endothelial cells are considered the primary target cells in the host, many non-endothelial cell types can also be efficiently parasitzied by pathogenic rickettsial species. Indeed, recent evidence using both R. conorii and R. rickettsii in murine models of fatal disease have shown that rickettsiae are found as intact bacilli in Kupffer cells and hepatocytes in the liver and in monocytes, neutrophils and lymphocytes within the hematogenous circulation. Interestingly, we have also demonstrated that two pathogenic species, R. conorii and R. rickettsii, are capable of proliferating within THP-1 macrophage-like cells in vitro while a non-human pathogen, R. montanensis, is quickly destroyed within structures containing markers of phago-lysosomes in these cells. These results strongly suggest that cell types other than endothelial cells likely play important roles in the progression of rickettsial disease and that the ability of rickettsiae to proliferate within phagocytes may be a distinguishing characteristic between virulent and non-virulent species. We have also recently determined that in the absence of neutralizing antibodies, R. conorii and R. rickettsii are able to evade complement-mediated killing in human and murine serum. These results demonstrated that rickettsial species likely have evolved active mechanisms to evade the bactericidal effects of complement deposition as the pathogen disseminates in the bloodstream to target organs and tissues. We have identified at least three rickettsial antigens, OmpB -peptide, RC1281/Adr1, and RC1282/Adr2 that are sufficient to mediate survival in normal human serum when expressed at the outer-membrane of a serum sensitive strain of E. coli. Homologues to these antigens exist in all pathogenic rickettsial species suggesting that the ability to actively perturb complement-mediated killing in the blood is a novel virulence attribute for this class of pathogens.

Abstract #140 Chlamydia trachomatis inclusion membrane proteins (Incs) are required for subversion of the host innate response and promote inclusion stability Mary M. Weber*, Jennifer Lam, Nicholas Noriea, Ted Hackstadt Host-Parasites Interactions Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT

Chlamydiae are obligate intracellular pathogens that replicate in a membrane bound compartment termed the inclusion. Early in the infection cycle the inclusion membrane is extensively modified by the incorporation of numerous type III secreted effector proteins. These inclusion membrane proteins (Incs) are incorporated such that they are exposed to the host cytosol and are poised to mediate crucial host-pathogen interactions. Inc proteins are characterized by a bi-lobed hydrophobic domain of 40 amino acids, a feature that has been used to predict 59 putative Incs. Until recently a lack of genetic tools significantly hampered our understanding of this important pathogen. However, recent advances, including the ability to generate site-specific mutants and to overexpress epitope-tagged proteins, make identification of bacterial virulence factors now feasible. Few Incs have known interacting host partners. Site-specific mutagenesis was used to generate targeted disruptions of 15 Incs of unknown function. Five of these mutants (CT229, IncC, CT288, CT383, and CT449) exhibited significant growth defects and formed smaller inclusions compared to the parental strain, supporting a role for these proteins in inclusion development. Premature lysis of the inclusion membrane followed by host cell rounding and detachment was evident for several of these mutants. Results obtained from Tunel assays, Annexin staining, and LDH release indicated that loss of CT229, IncC, or CT383 stimulated programed cell death pathways. By 18h post-infection, caspase and Poly (ADP-ribose) polymerase (Parp) cleavage was observed in cells infected with these mutants. Collectively

92 these results implicate several inclusion membrane proteins in promoting stability of the inclusion membrane, thereby preventing host cell death, potentially by sequestering the bacteria from the cytosol and avoiding activation of the innate immune response.

Abstract #141 Elucidation of the Biochemical Interaction between Rickettsia conorii Adr1 and Human Host Vitronectin A.I. Fish*, S.P. Riley, J.J. Martinez Vector-borne Diseases Laboratory, Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 70803

Spotted fever group Rickettsia sp. are injected directly into the mammalian bloodstream by hematophagous arthropods. Once in the bloodstream and during dissemination, the survival of these pathogens is dependent upon the ability of these bacteria to evade innate host defenses until a proper cellular host is reached. Rickettsia conorii expresses an outer membrane protein, Adr1, which binds the complement regulatory protein vitronectin to promote resistance to the anti- bacterial effects of the complement system. Homologues of Adr1 are present in all sequenced pathogenic rickettsial species suggesting that the ability to avoid serum-mediated killing is a conserved attribute for this class of bacteria. Adr1 is predicted to consist of 8 transmembrane beta sheets that form a membrane spanning barrel with 4 peptide loops exposed to the extracellular environment. We have previously demonstrated that Adr1 derivatives containing either loop 3 or 4 are sufficient to bind vitronectin and mediate resistance to serum killing when expressed at the outer-membrane of E. coli. To further characterize this favorable protein-protein interaction, I have utilized co-sedimentation to demonstrate a salt-sensitive heparin-independent interaction between Adr1 and human vitronectin. By utilizing various truncated vitronectin peptides, I have also mapped the protein-protein interaction to the C-terminal region of vitronectin. Additionally, we have utilized site-directed mutagenesis to determine the specific amino acid residues located within loops 3 and 4 that mediate the Adr1/vitronectin interaction. Our ultimate goal for the project is to fine map the interacting regions of vitronectin and Adr1, with the expectation of using this information to define the roles of Adr1 and vitronectin in rickettsial pathogenesis.

Abstract #142 Orientia tsutsugamushi effector Ank9 targets host cell secretion pathways via Golgi/ER localization and COPB2 interaction Andrea R. Beyer*, Kyle G. Rodino, Lauren VieBrock, Ryan S. Green, Jason A. Carlyon Microbiology & Immunology, Virginia Commonwealth University, School of Medicine, Richmond, VA

Orientia tsutsugamushi causes scrub typhus, an emerging and potentially fatal infection that afflicts one million people annually. The host cell processes co-opted by this obligate intracellular bacterium are poorly defined. Among microbes, O. tsutsugamushi boasts one of the largest arsenals of ankyrin repeat-containing proteins (Anks), secreted effectors capable of targeting and manipulating host cell pathways. We have focused on characterizing a subset of 20 Anks, many of which possess a C-terminal F-box involved in exploiting host ubiquitination machinery. Additionally, fourteen Anks localize to the host endoplasmic reticulum (ER) by uncharacterized mechanisms. One such ER-localizing, F-box-containing effector, Ank9, was found to also exhibit accumulation in the Golgi. A Golgi localization domain (GLD), mimicking the GRIP domain of eukaryotic golgins, was mapped to the Ank9 N-terminus and found to be necessary and sufficient for Golgi accumulation. Ank9 does not traffic to the ER without first going to the Golgi. However, Ank9 localization is not altered with the retrograde trafficking inhibitor Brefeldin A, suggesting an alternate Golgi-to-ER route. COPB2, a subunit of the COPI coatomer complex which mediates Golgi-to-ER trafficking, was identified and validated as an Ank9 binding partner. Furthermore, ectopically expressed Ank9 destabilized the Golgi and ER. With its implication in disrupting host secretory pathways, we examined the effect of Ank9 on induction of ER stress and host protein secretion. Ank9 induced expression of the unfolded protein response transcription factor, ATF4, and inhibited secretion of a luciferase reporter in a GLD-, but not F-box-, dependent manner. This report identifies a novel microbial GLD as the first motif by which any Orientia effector targets the secretory pathway, and evidences how a single bacterial effector uses eukaryotic-like functional domains to co-opt multiple host processes.

93 Abstract #143 Efficiencies and optimization of targeted insertional mutagenesis using a group II intron in Rickettsia rickettsii Nicholas F. Noriea*, Tina R. Clark, and Ted Hackstadt Host-Parasite Interactions Section, Laboratory of Intracellular Parasites, Rocky Mountains Labs, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840

Genetic tools to create targeted gene disruptions in Rickettsia rickettsii are limited due to the obligate intracellular nature of the bacterium and lack of suitable culture media. Previously we reported the creation of an isogenic mutant in the highly virulent strain, Sheila Smith, by insertion of intronic RNA into the open reading frame of ompA via an LtrA group II intron retrohoming system. Group II introns are mobile genetic elements that have the ability to insert into specific DNA sites. The ability for group II introns to splice and insert is dependent on many variables including the intronic RNA structure, transcript level, and size. Here we examine several modifications to the group II intron Rickettsial suicide vector, pARR, to assess efficiency in insertion of intronic RNA into targeted DNA sites. Overall, efficiency was most dependent on the orientation of the selectable marker on the intron coding sequence. Due to limitations in available antibiotics appropriate for research in R. rickettsii, two effective bacteriostatic antibiotics Rifampin and Spectinomycin, were tested as potential selectable markers. While retrohoming is size-dependent in regards to the intronic RNA, no difference in intronic insertion efficiency was detected using the smaller rifampin resistance gene (453bp) versus the larger Spectinomycin resistance gene (1017bp). pARR is a suicide vector and thus must express the intronic pre-RNA, holoenzyme, and selectable marker within several hours of transformation. Promoter strength driving transcription of the intronic pre-mRNA as well as the selectable marker could play an important role Rickettsial intron retrohoming efficiency. Rickettsial promoters of several constitutively expressed genes (ompA, ompB, gltA, trmD, rpsL) were examined for promoter strength in both E. coli and R. rickettsii. Promoters of various strength were then tested both as drivers of intronic RNA and antibiotic resistance cassette transcripts. Somewhat counterintuitively, the addition of a rickettsial promoter onto the selectable marker had a negligible effect on antibiotic-resistant Rickettsial clones but did increase the number of false-positive insertional events. The ability to create isogenic mutants in R. rickettsii is a novel and powerful tool in research. Optimizing this tool provides great value in the reduction of resources necessary to create isogenic Rickettsial mutants.

Abstract #144 The first crystal structure of the soluble domain of RC1339/APRc from R. conorii reveals a striking conservation with the typical fold of retropepsins Mi Li1,2, Alla Gustchina1, Pedro Curto3,4, Rui Cruz3,5, Marisa Simões3,5, Carlos Faro3,5, Juan J. Martinez4, Alexander Wlodawer1 and Isaura Simões3,4,5* 1Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD, USA 2Basic Science Program, Leidos Biomedical Research, Frederick Nat’l Laboratory for Cancer Research, Frederick, MD 3CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal 4Vector-Borne Diseases Laboratories, Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA 5Biocant, Biotechnology Innovation Center, 3060-197 Cantanhede, Portugal

The common secondary structure template among domains/monomers of pepsin-like aspartic proteases and retropepsins supports the view that these proteases are evolutionarily related, and that pepsins may have arisen by gene duplication and fusion of an ancestral form of retropepsins. The nature of this primordial single-lobed aspartic protease has been the matter of debate over the years, mostly due to the lack of compelling evidence for the presence of these enzymes in prokaryotes. Recently, we reported the identification of a gene coding for a membrane-embedded, single-lobed aspartic protease, highly conserved in the genomes of 55 species of Rickettsia. Using Rickettsia conorii gene homolog rc1339, we provided evidence that the encoded product (APRc), indeed shares several enzymatic properties with viral retropepsins (Cruz, et al., PLoS Pathog., 2014). These resemblance of enzymatic features suggested that APRc might indeed represent a more primordial form of retropepsins. In this work, crystal structures of two constructs of the soluble domain of APRc from Rickettsia conorii were determined in three different crystal forms. The results clearly show that the fold of APRc monomer resembles that of viral retropepsins, but the quaternary structure of the dimer differed from the canonical

94 retropepsins. Whereas the observed dimer is most likely an artefact of expression/crystallization since it cannot support the reported enzymatic activity of APRc, the fold of the core of each monomer is very closely related to the fold of retropepsins. Overall, our results support the concepts that APRc may indeed represent a putative common ancestor of monomeric and dimeric aspartic proteases, as well as possible existence of a different evolutionary pathway for these enzymes. Moreover, our recent observations showed that treatment of R. conorii-infected mammalian cell cultures with indinavir (a HIV-1 protease inhibitor) result in a dramatic decrease in the ability of R. conorii to bind to and subsequently proliferate within these cells, further pointing towards a key role of APRc in a proteolytic pathway relevant to the rickettsial life cycle.

Abstract #145 Structural characterization of the Anaplasma marginale antigenically variant Msp2 protein Telmo Graça*1,2, Marta G. Silva2, Alla S. Kostyukova3 and Guy H. Palmer1,2 The Paul G. Allen School for Global Animal Health1, Department of Veterinary Microbiology and Pathology2, School of Chemical Engineering and Bioengineering3, Washington State University, Pullman, Washington 99164

Antigenically variant gram negative bacteria use recombinatorial mechanisms to encode new outer membrane protein variants in order to infect and persist in susceptible mammalian hosts. Anaplasma marginale infection results in lifelong persistence and is linked to sequential expression of unique Hypervariable Regions (HVR) of the Major Surface Protein 2 (Msp2). The generation of new antigenic variants at the HVR locus is the result of a mechanism of unidirectional homologous recombination where a limited set of non-functional donor Msp2 alleles are translocated - as a whole or in oligonucleotide segments - into the single msp2 expression site. Interestingly, although Msp2 is immunodominant, the generated HVR-specific antibody is transient and allows re-emergence of similar variants at later timepoints in infection. Using circular dichroism, complemented with structural modeling, we determined that Msp2 is mostly composed of random coils. The N- and C-terminals alternate random coils with β-sheets and the central HVR is predominantly characterized by random coils anchored by α-sheets at the edges. We propose that the predominance of random coils in the HVR generates a flexible surface exposed structure and that this flexibility results in the short lived antibodies observed during infection.

Abstract #146 Immunity to Infections Caused by Organisms of the Genus Rickettsia David H Walker* and Rong Fang Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555

The initial target cells of Rickettsia in skin are mononuclear phagocytes (i.e., macrophages and/or dendritic cells). Rickettsial infection of dendritic cells results in intraphagosomal and cytosolic rickettsiae, enabling antigen presentation by the MHC class I and class II pathways, respectively, and induces full maturation of dendritic cells with high-level expression of MHC II and co-stimulatory molecules, production of IL-12-p 40, and priming CD4 T cells. Adoptive transfer of rickettsiae- infected dendritic cells into mouse footpads 24 hours prior to an ordinarily lethal rickettsial challenge confers immune protection and results in survival associated with enhanced NK cell cytotoxic activity and IFN-ɣ production. Dendritic cells are activated by rickettsiae through TLR 4, and MyD88 plays a key role in signal transduction that mediates immunity to rickettsiae. Activation of inflammasome also plays an important role in immunity to rickettsiae in which ASC is crucial. Systemic rickettsial infection involves primarily endothelial cells and secondarily macrophages. Experiments with gene knockout mice and antibody-mediated depletion revealed the important contributions of CD8 T cells, MHC-I, and perforin, emphasizing the critical role of CTL in clearance of infection. Antibodies do not contribute to recovery from a primary infection as they appear only after recovery has begun. Intracellular rickettsiacidal activity is stimulated by IFN-ɣ,TNF-α,IL-1β, and RANTES and is mediated by production of NO and reactive oxygen species in human endothelial cells and by IDO limitation of tryptophan stores in human macrophages. Rickettsial killing in cytokine-activated endothelial cells mediated by NO is associated with autophagy and autophagosomal digestion of rickettsiae. Depletion of IFN-ɣ or TNF-α converts an ordinarily sublethal rickettsial infection to a lethal outcome, and IFN-ɣ knockout mice are highly susceptible to ordinarily sublethal doses of rickettsiae. Previous infection confers solid immunity to subsequent homologous infections; cross protection between the spotted fever and typhus groups is mediated by T cells.Adoptive 95 transfer of immune CD4 and CD8 T cells and antibodies to OmpA and OmpB are protective against a lethal rickettsial challenge, indicating that these would be effective mediators of vaccine-induced immunity.

Abstract #147 Innate immune control of Coxiella burnetii infection within macrophages William P. Bradley1,2, Mark A. Boyer2, Hieu T. Nguyen2, L. Dillon Birdwell1,2, Janet Yu2, Juliana M. Ribeiro3, Susan R. Weiss2, Dario S. Zamboni3, Craig R. Roy4, and Sunny Shin1,2.# #Corresponding author, e-mail: [email protected] 1Cell and Molecular Biology Graduate Group, Perelman School of Medicine, Univ. of Pennsylvania, Philadelphia, USA 2Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA 3Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil 4Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA

Coxiella burnetii replicates within permissive host cells by employing a Dot/Icm type IV secretion system (T4SS) to translocate bacterial effectors that promote its intracellular survival. C57BL/6 mouse macrophages restrict intracellular replication of the C. burnetii Nine Mile phase II (NMII) strain. However, eliminating Toll-like receptor (TLR) 2 permits bacterial replication, indicating that bacterial restriction is immune-mediated. Thus, we examined whether additional innate immune pathways are employed by C57BL/6 macrophages to sense and restrict NMII replication. Although C. burnetii NMII uses its T4SS to translocate bacterial products into the cytosol of C57BL/6 macrophages, there was little evidence of cytosolic immune sensing of NMII, as there was a lack of inflammasome activation, T4SS-dependent cytokine responses, and type I IFN production, nor were these pathways required to restrict bacterial replication. Instead, endogenous TNF produced upon TLR sensing of C. burnetii NMII was required to control bacterial replication. Therefore, our findings indicate a primary role for TNF produced upon TLR-mediated detection of C. burnetii NMII, rather than cytosolic immune sensing, in enabling C57BL/6 macrophages to restrict C. burnetii infection.

Abstract #148 Investigating the Roles of T cell-mediated Immunity during Scrub Typhus Guang Xu1*, Thomas R. Shelite2, Nicole L. Mendell1, Lynn Soong2, Donald H. Bouyer1, David H. Walker1* 1Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555, United States 2Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, Texas 77555, United States

Scrub typhus, caused by a Gram-negative obligately intracellular coccobacillus, Orientia tsutsugamushi, is a long neglected but important tropical disease. O. tsutsugamushi causes illness in one million people each year, and 1 billion people are at risk. Without appropriate diagnosis and treatment, the disease can cause severe multiorgan failure with a case fatality rate of 7-15%. The current gaps include the unknown mechanisms behind the interactions between O. tsutsugamushi and host immunity. Using intravenous (i.v.) mouse model developed by our laboratory, we determined that more CD8+ T cells + than CD4 T cells appeared in the spleen of infected mice after 12 dpi. We also found that Treg cells and the proportion of T cells producing IL-10 were significantly increased from 6 dpi, which was in parallel with the body weight loss and increased bacterial loads. We further used CD8-/- mice, MHC I-/- mice and wild type (WT) C57BL/6 control counterparts to determine the importance of CD8+ T cells as well as MHC I molecules. After being administered an ordinarily sub-lethal dose of O. tsutsugamushi, most CD8-/- mice expired by 13 dpi except 1 that expired on 15 dpi, while all WT mice survived. All MHC I-/- mice that received the same Orientia dose expired by 12 dpi. Bacterial loads in the lung, kidney, liver and spleen of CD8-/- mice and MHC I-/- mice were significantly higher than those in WT mice. IFN-γ mRNA levels in the lung of CD8-/- mice were significantly higher than in WT mice. There was significantly less CXCL-9 in the lung of MHC I-/- mice than in the WT mice. There was no statistically significant difference in the mRNA levels of IL-10 in the 4 organs among CD8-/- mice, MHC I-/- mice and WT mice. We also found a greater pro-inflammatory immune response in the tissues of WT mice than in the two types of KO mice. More apoptosis were observed in the liver of MHC I-/- mice than in the WT mice. More studies are necessary to better understand the role of host immunity during Orientia infection.

96 Abstract #149 Immunoinhibitory receptors PD-1 and LAG-3 contribute to T-cell exhaustion during Anaplasma marginale infection Tomohiro Okagawa1, Satoru Konnai1, James R. Deringer2, Massaro Ueti3, Glen A. Scoles3, Shiro Murata1, Kazuhiko Ohashi1, and Wendy C. Brown2* 1Graduate School of Veterinary Medicine, Hokkaido University, Sapporo Japan; 2College of Veterinary Medicine, Washington State University, Pullman, WA; 3Animal Disease Research Unit, ARS, USDA, Pullman, WA

Several published studies from our lab have documented the induction of an exhausted CD4 T-cell response, specific for Anaplasma marginale antigens induced by prior immunization, following either needle or tick-borne infection with this bacterial pathogen. This exhausted phenotype, characterized by significantly reduced or absent A. marginale-specific T- cell proliferation and cytokine production and T-cell deletion, was partially reversed when the persistent infection was cleared by tetracycline therapy, supporting a mechanistic role of high antigen load in suppression. Furthermore, induction of T-cell exhaustion required the presence of the specific priming T-cell epitope on the infecting bacteria, suggesting T-cell receptor engagement by the immunization-primed T cells was involved. Herein we show that induction of T cell immunoinhibitory receptors programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) are upregulated on all T cell subsets following infection, with highest percentages of PD-1+ LAG-3+ double positive T cells occurring at the peak of infection, concurrent with the rapid loss of A. marginale-specific CD4 T cell responses. The ligand PD-L1 was also significantly upregulated on CD14+ antigen presenting cells, following the same kinetics of expression. Finally, in vitro antibody blockade of dual PD-1–PD-L1 and LAG-3–MHC class II receptor-ligand interactions resulted in partial, but significant, restoration of the antigen-specific T-cell response. These results support the contribution of PD-1 and LAG-3 receptor-ligand interactions in inhibiting the A. marginale antigen induced-specific CD4 T-cell recall response following A. marginale infection in cattle.

Abstract #150 Differences in the Intracellular Fate of Two Spotted Fever Group Rickettsia in Macrophage-Like Cells Pedro Curto1,2,3*, Isaura Simões3,4,5, Sean Riley5, Juan J. Martinez5 1PhD Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, Univ. of Coimbra, Portugal; 2Institute for Interdisciplinary Research, Univ. of Coimbra, Portugal. 3CNC-Center for Neuroscience and Cell Biology, Coimbra, Portugal 4Biocant, Biotechnology Innovation Center, Cantanhede, Portugal 5Vector Borne Disease Laboratories, Department of Pathobiological Sciences, LSU School of Veterinary Medicine, Baton Rouge, LA, USA

Spotted fever group (SFG) Rickettsia are recognized as important agents of emerging human tick-borne diseases worldwide such as Mediterranean spotted fever (R. conorii) and Rocky Mountain spotted fever (R. rickettsii). Recent studies in several animal models have provided evidence of non-endothelial parasitism by different Rickettsia species suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in the pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. We evaluated the ability of two SFG Rickettsia species, R. conorii (a recognized human pathogen) and R. montanensis (a non-virulent member of SFG), to proliferate in THP-1 macrophage- like cells and also within a non-phagocytic cell line, Vero. Growth dynamics were evaluated by quantitative PCR and the ability of the two species to associate with and invade into THP-1 and Vero cells was evaluated by immunofluorescence microscopy-based assays. Our results demonstrated that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in Vero cells, but was drastically compromised in the ability to proliferate within THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the remaining bacteria did not appear to be affected. Our results also demonstrated that the few R. montanensis that could invade the THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2, a lysosomal marker. In contrast, R. conorii was present as intact bacilli and free in the cytoplasm in both cell types. These findings suggest that a major phenotypic difference between a non- pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells and may provide an explanation as to why certain SFG rickettsial species are not associated with disease.

97 Abstract #151 Orientia tsutsugamushi ankyrin repeat-containing proteins Ank1 and Ank6 inhibit the host NF-κB proinflammatory pathway Sean M. Evans* and Jason A. Carlyon Dep’t of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA

Orientia tsutsugamushi is an obligate intracellular bacterium that infects human leukocytes to cause scrub typhus, a potentially fatal disease that infects over one million persons annually in the Asia-Pacific region. To better understand why these innate immune cells are unable to eliminate O. tsutsugamushi, we addressed the activity of host NF-κB, an essential and quick responding proinflammatory transcription factor. During cellular quiescence, IκBα, an ankyrin repeat-containing protein (Ank), sequesters NF-κB in the cytosol through non-covalent interactions via its ankyrin repeats. In the presence of a stress-induced signal (e.g. TNF-α), NF-κB is liberated from IκBα, thereby allowing NF-κB to translocate to the nucleus to act as a transcription factor. Screening of O. tsutsugamushi infected cells at an MOI of 1 over a 24-hour period revealed inhibition of NF-κB nuclear accumulation as early as 8 hours. When infected host cells are stimulated with TNF-α, IκBα degradation occurs as expected, but nonetheless NF-κB dependent gene transcription remains downregulated. This in turn led us to examine possible bacterial effectors that could prevent NF-κB nuclear translocation subsequent of IκBα degradation. In silico analyses led us to pursue two confirmed O. tsutsugamushi type 1 secretion system effectors, Ank1 and Ank6, each of which carry ankyrin repeats that exhibit homology to two tandemly-arranged ankyrin repeats of IκBα. We investigated whether or not these Anks are capable of altering NF-κB function. Immunofluorescence microscopic analysis of TNF-α treated cells ectopically expressing Ank1 or Ank6 showed a significant decrease in NF-κB nuclear accumulation. Additionally, these Anks also significantly inhibited NF-κB dependent gene transcription. Finally, immunoprecipitation experiments confirmed that Ank1 and Ank6 are capable of interacting with NF-κB. These data implicate that these two Anks likely play roles in O. tsutsugamushi infection by binding NF-κB to prevent its nuclear accumulation, which results in the downregulation of the NF-κB-driven innate immune response.

Abstract #152 Rickettsiologist Paul F. Zdrodovskyi - larger than life and not just for his famous book Marina E. Eremeeva1, Stanislav N. Shpynov2, Irina V. Tarasevich2 1Jiann-Ping Hsu College of Public Health, Georgia Southern University, Statesboro, GA; 2N.F. Gamaleya FRCEM, Moscow, Russia

Russian rickettsiologist Paul F. Zdrodovskyi is known to our mentors in rickettsiology from his famous book titled “The Rickettsial Diseases”. It was the first seminal Russian book on rickettsiology which was translated in English in 1960, and it was published at the peak of his scientific career. 2015 was the 125th anniversary of his birth. The purpose of this presentation is to honor this great rickettsiologist by highlighting Zdrodovskyi’ biography and scientific accomplishments for this generation of rickettsiologists as this detailed work still includes much information of contemporary interest. After receiving his primary education in a Seminary, Zdrodovskyi graduated from Medical Faculty of the Kazan University. Starting as a recruit in the Russian Army, his life was an example of service to the profession refracted through the prism of the dramatic history of Russia in the 20th century. His career evolved from his time as an Army epidemiologist to elevation to full member of the Russian Academy of Medical Sciences and the Head of the Department of Epidemic Typhus and Other Rickettsioses at the famous Gamaleya Research Institute in Moscow. The geography of his assignments extended from Azerbaijan to St. Petersburg and Moscow; the topics of his work encompassed control of malaria to diphtheria and tetanus vaccination; his contributions include vaccine development for Q fever and epidemic typhus, and establishing standards for the specific prophylaxis of all the rickettsial diseases known in the former USSR. He published in Russian, English and French. His bibliography includes over 30 monographs and 500 papers from clinical infection to immune responses to zoonotic diseases; many of these publications are still relevant as they summarize very fundamental information in the fields of his expertise and provided the basis for education of two whole generations of very productive rickettsiologists until the fall of the USSR. Those individuals in turn shared their expertise and experience with the western rickettsiologists through the international rickettsial meetings held from 1968 to 1996 in the former Czechoslovakia.

98 Abstract #153 In depth analysis of Anaplasma marginale: what we have learned with from deep sequencing Kelly A. Brayton* Program in Genomics, Dep’t of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA

Anaplasma marginale is an obligate intracellular pathogen of cattle with a global distribution. The organism was first described by Sir Arnold Theiler in the early 1900s, but molecular understanding was hard fought for this organism that could only be grown in the bovine host until the mid 1990s when it was also cultured in tick cells. A century after its description the first genome sequence for A. marginale was generated in 2005, revealing much that was previously unexplored about this pathogen. Of particular note, the genome provided insights into the mechanism of antigenic variation and allowed us to determine that a gene conversion mechanism was employed to effect immune evasion. Also, a previously undetected type four secretion system was found. Subsequent genome sequences have allowed comparative genomics analyses which have identified genes implicated in tick transmission. To capitalize on these comparative analyses, we have generated some of the most robust high throughput transcriptomic analyses undertaken for obligate intracellular organisms which allowed comparisons between transmissible and non-transmissible strains as well as a transmissible strain grown in the bovine host and grown in tick cells. Finally, we have determined the strand specific transcriptome of the St. Maries strain of A. marginale and see that is different when isolated from tick cells vs. bovine cells. Antisense RNA transcription is prevalent in A. marginale and provides a mechanism for gene regulation in this pathogen through modulation of protein translation. This mechanism of gene regulation is employed by pathogens that have to compensate for less flexible genomes and have fewer opportunities for genetic exchange in the face of changing environments.

Abstract #154 Using Genomics and Genetics to Unravel Phase Variation in Coxiella burnetii Paul A. Beare* and Robert Heinzen Coxiella Pathogenesis section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT

Axenic growth of Coxiella burnetii dramatically enabled development of genetic tools that are now being used to define pathogen virulence determinants. An intriguing question in C. burnetii biology relates to mechanisms of phase variation wherein virulent phase I bacteria, producing a full-length LPS, convert to avirulent phase II bacteria, producing severely truncated LPS, following serial ex vivo passage. Strains producing an intermediate length LPS termed Crazy have also been described. A direct correlation between LPS length and virulence has been established with the isogenic strains Nine Mile phase I (NMI) fully virulent), Nine Mile Crazy (NMC; moderately virulent), and Nine Mile phase II (NMII; avirulent). NMII contains a large chromosomal deletion eliminating 21 ORFs predicted to function in O-antigen biosynthesis. However, NMC contains a larger deletion and some phase II strains lack a deletion, indicating this genetic lesion per se is not responsible for phase II conversion. Using monoclonal antibodies specific to LPS chemotypes and serial passage, we show that phase I to phase II transition occurs similarly within strains of different genomic groups. Passage of clonal NMC also results in generation of phase II organisms. Genome sequencing of several C. burnetii isolates revealed mutations potentially associated with LPS modification and/or phase conversion. Targeted inactivation of cbu0678 confirmed its role in generating the Crazy LPS chemotype. Complementation of cbu1657 in the Australian QD phase II strain confirmed its role in generating an antigenically-distinct phase II LPS. Both CBU0678 and CBU1657 are predicted to be involved in heptose modification. Collectively, our results reveal mutation of a single gene is responsible for conversion of phase I to Crazy LPS, and that the large deletion is a rare event that follows this conversion. However, the genetic lesion that accounts for the severe LPS truncation of phase II C. burnetii remains elusive.

Abstract #155 Genomic characterization of Orientia tsutsugamushi isolates from Southeast Asia, the Pacific Islands, Australia, Japan, and Korea Kristin E. Mullins*1, Amy Fleshman2, Crystal M. Hepp2, Jason, Sahl2, Daniel Paris3, 4, Sabine Dittrich3, 5, 6, Paul Newton3, 5, Talima R. Pearson2, Allen L. Richards1, 7 99 1Naval Medical Research Center, Silver Spring, MD, United States; 2Northern Arizona University, Flagstaff, AZ, USA; 3Centre for Tropical Medicine, University of Oxford, Oxford, England, UK; 4Mahidol-Oxford Tropical Medicine Research Unit, Bangkok, Thailand; 5Wellcome Trust–Mahosot Hospital–Oxford Tropical Medicine Research Collaboration, Vientiane, Lao People's Democratic Republic; 6Foundation for Innovative New Diagnostics, Geneva, Switzerland; 7Uniformed Services University of the Health Sciences, Bethesda, MD, USA

The obligate intracellular, Gram-negative bacterium Orientia tsutsugamushi is transmitted by the bite of infected chiggers (Leptotrombidium spp) and is the causative agent of scrub typhus. O. tsutsugamushi infections have been documented throughout Southeast Asia, the southwestern Pacific Islands, Korea, parts of Russia, China, Japan, Australia, New Zealand, Pakistan, and India, causing a wide range of disease presentations. Disease can range from mild or asymptomatic infections to extremely severe disease such as meningitis, encephalitis, and shock, which in some cases can lead to death. The genus Orientia contains only two known species, O. tsutsugamushi and O. chuto, however, the former is extremely genetically diverse with greater than 100 known O. tsutsugamushi genotypes, yet the genetic diversity of O. tsutsugamushi is poorly understood. The most widely used typing system employs a single, highly variable gene, the 56-kDa type-specific antigen gene, which, most likely, does not provide a true picture as to the genetic relationships between O. tsutsugamushi isolates or their disease presentations. In an attempt to gain a better understanding of the genetic diversity, genome evolution, and variation in disease severity seen with O. tsutsugamushi, genome sequencing was applied to 32 isolates from diverse locations throughout the endemic area. Comparisons between the 32 isolates and 4 previously sequenced O. tsutsugamushi isolates were made at the whole genome, protein, and single gene levels. In addition to full genome analyses, suspected virulence determinants were analyzed for the 40 isolates and for 65 clinical isolates from scrub typhus patients in Laos who presented with varying disease severity. Analyses revealed characteristics that have not previously been documented for any species. These include the complete lack of a “clonal frame” due to extensive homologous recombination among genomes clouding the ability to determine evolutionary relationships. Also, the high genetic diversity among genomes appears to be driven not by importation of exogenous DNA, but rather by divergence and decay of existing genes. These analyses provide a clearer picture of the genetic diversity seen with O. tsutsugamushi and the genetic relationships between O. tsutsugamushi isolates that could not be elucidated using current typing schemes or by analyses of single genomes. Further investigations need to be undertaken to develop valid typing schemes and to determine the full extent to which the genetic diversity seen in O. tsutsugamushi influences the wide array of disease presentations seen across the endemic areas.

Abstract #156 Biostatistical prediction of genes essential for growth of Anaplasma phagocytophilum in a human promyelocytic cell line using a random transposon mutant library Kate O’Conor* and Ulrike G. Munderloh College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108

Background: Anaplasma phagocytophilum (Ap) is an obligate intracellular tick-borne zoonotic bacterium that is expanding its range in North America and Europe. Ap is the agent of human anaplasmosis, the second most common tick-associated illness after Lyme disease in the US. Even though its small genome of 1.5 Mb has been sequenced, about 40 % of the genes it encodes are hypothetical and have no known function. A mutant library of Ap was previously generated using the Himar1 transposon system to insert genes for a red fluorescent protein (mCherry) and resistance to the antibiotic spectinomycin randomly into the Ap genome. Although the transposons were likely randomly inserted, only invasion- and replication- competent bacteria were able to grow, thus genes without insertions are potentially essential for growth in human promyelocyte HL-60 cells. However, the density of recovered mutants was nonsaturating, meaning that some genes were spared randomly rather than because they were essential for growth in this cell line. A biostatistical method was used to identify essential genes in a subsaturated library of Mycobacterium tuberculosis Himar1 transposon mutants, and we applied this method to our Ap library. Results: Transposon mutagenesis produced 857 Ap isolates, and insertion sites were mapped back to the genome using Illumina sequencing of pooled transformant DNA, which identified approximately 1100 insertions. Insertion sites were distributed non-randomly, notably sparing some genes and operons. Since the Himar1 transposon is inserted randomly into the genome at a site reading TA, the number of potential insertion sites in each gene as well as the number of viable mutants with transposon insertions within each gene can be used in a Markov chain Monte Carlo model to estimate the probability that a given gene is essential for the organism. Genes predicted to be essential 100 for survival in HL-60 cells include metabolism and housekeeping genes, components of the type IV secretion system, and antioxidant defense molecules as well as several hypothetical proteins. Conclusion: We have identified a number of genes predicted to be essential for invasion and replication of Ap in HL-60 cells. This method will help target genes for further investigation to elucidate their role in Ap pathogenesis.

Abstract #157 Analysis of Complete Genome Sequence and Major Surface Antigens of Neorickettsia helminthoeca, Causative Agent of Salmon Poisoning Disease Mingqun Lin *1, Katherine Bachman 1, Zhihui Cheng 1, Sean C. Daugherty 2, S. Nagaraj 2, Naomi Sengamalay 2, S. Ott 2, A. Godinez 2, Luke Tallon 2, Lisa Sadzewicz 2, Sushma Parankush 2, Claire Fraser 2,3, Julie Dunning Hotopp 2,4, Yasuko Rikihisa 1 1 Dep’t of Veterinary Biosciences, The Ohio State University, Columbus, OH, USA;n2 Institute for Genome Sciences, 3 Department of Medicine, 4 Dep;t of Microbiology and Immunology, University of Maryland School of Medicine, MD, USA

Neorickettsia helminthoeca is an obligatory intracellular bacterium of digenetic trematodes that causes Salmon poisoning disease (SPD) in domestic and wild canids, an acute and often-fatal illness. The bacterium is transmitted from trematodes to dog monocytes or macrophages when dogs eat salmonid fish containing encysted trematodes infected with N. helminthoeca. In this study, we analyzed the complete genome sequence and major antigens of this organism. The genome of N. helminthoeca Oregon consists of a small, single circular chromosome of 884,232 bp that encodes 774 proteins. Although N. helminthoeca has a very limited capacity to synthesize amino acids and lacks many metabolic pathways, it is capable of producing all major vitamins, cofactors, and nucleotides, which may be beneficial to the trematode host. Like other members of the family Anaplasmataceae, N. helminthoeca lacks genes for lipopolysaccharide biosynthesis. However, it encodes complete pathways to synthesize peptidoglycan, suggesting its mechanical strength and inflammatory potential. Genes potentially involved in the pathogenesis of N. helminthoeca were identified, including two-component regulatory systems, type I and IV secretion systems, and putative transcriptional regulators. Five predicted major surface antigens of N. helminthoeca, including P51, NSP-1/2/3, and SSA, were cloned and expressed. Western blotting data showed that all these proteins were recognized by experimentally infected dog blood specimens, whereas SSA was reacted strongest by naturally infected SPD dog sera. These findings facilitate our understanding of the biology and pathogenesis of this elusive environmental bacterium, and provide the tools with which to design rapid and sensitive serodiagnostic methods and new prevention strategies for SPD.

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ASR 2016 Travel Award Recipients

John Beckmann Yale University Kevin Bewley Public Health England Chelsea Cockburn Virginia Commonwealth University Francy Crosby University of Florida Pedro Curto University of Coimbra / Louisiana State Univ. Sean Evans Virginia Commonwealth University Alison Fedrow NMRC/HJF/Shippensburg University Abigail Fish Louisiana State University Cory Gall University of Pretoria Maria Galletti University of Sao Paulo Telmo Graça Washington State University Crystal Hepp Northern Arizona University Na Jia Beijing Institute of Microbiology & Epidemiology Agatha Kolo University of Pretoria Munegowda Koralur Manipal University Eleanor Latomanski The University of Melbourne Jung Keun Lee Mississippi State University Taslima Lina University of Texas Medical Branch Alice Maina Naval Medical Research Center Jodi McGill Kansas State University Kristin Mullins Naval Medical Research Center Girish Neelakanta Old Dominion University Kate O’Conor University of Minnesota Rahul Raghavan Portland State University Dana Shaw University of Maryland Sunny Shin University of Pennsylvania Lynn Soong University of Texas Medical Branch Vaughan Trounson Victoria University Ying Wang Kansas State University

102 Caylin Winchell University of Arkansas for Medical Sciences Guang Xu University of Texas Medical Branch

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The American Society for Rickettsiology would like to thank our sponsors:

Funding for this conference was made possible [in part] by R13 AI126727- 01 from the National Institute of Allergy and Infectious Diseases. The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the U.S. Department of Health and Human Services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government.

104 Index of Lead Presenters

Name Email Abstract # Abdullayev, Mirfazil [email protected] 25 Allerdice, Michelle [email protected] 18 Ayres, Bryan [email protected] 88 B. Saito, Tais [email protected] 40 Battisti, James [email protected] 35, 130 Beare, Paul [email protected] 153 Bechelli, Jeremy [email protected] 63 Beckmann, John F. [email protected] 77 Benach, Jorge [email protected] Historical Lecture Beyer, Andrea [email protected] 142 Brayton, Kelly [email protected] 152 Brown, Wendy [email protected] 149 Chae, Joon-Seok [email protected] 123 Chao, Chien-Chung [email protected] 127 Chen, Hua-Wei [email protected] 120 Ching, Wei-Mei [email protected] 118 Choi, Kyoung-Seong [email protected] 17, 87 Chowdhury, Imran [email protected] 28 Cockburn, Chelsea [email protected] 68 Cockrell, Diane C. [email protected] 54 Crocquet-Valdes, Patricia [email protected] 45 Crosby, Francy [email protected] 6, 134 Curto, Pedro [email protected] 150 Dasch, Greg [email protected] 16, 102 Dumler, J. Stephen [email protected] 81 Dzul-Rosado, Karla [email protected] 126, 21 Eremeeva, Marina [email protected] 151 Ericson, Marna [email protected] 12 Evans, Sean [email protected] 150 Farris, Tierra [email protected] 57, 109 Fikrig, Erol [email protected] Memorial Lecture Fish, Abigail [email protected] 141 Fitzpatrick, Kelly [email protected] 49 Fleshman, Amy [email protected] 23 Frangoulidis, Dimitrios [email protected] 14 Funk, Joel [email protected] 52 Gall, Cory [email protected] 22 Galletti, Maria [email protected] 43 Ganta, Roman [email protected] 69, 136 Garza, Daniel [email protected] 61 Gaywe, Jariyanart [email protected] 112, Gilk, Stacey [email protected] 9 Gong, Bin [email protected] 158 Graca, Telmo [email protected] 145 105 Healy, Sean [email protected] 44 Hwang, Seon Do [email protected] 124 Ismayilova, Rita [email protected] 98 Jakkula, Laxmi U.M.R. [email protected] 27 Jia, Na [email protected] 85 Jiang, Ju [email protected] 24 Jirakanwisal, Krit [email protected] 113 Karpathy, Sandor E. [email protected] 96 Kato, Cecilia [email protected] 86, 122, 129 Clark, Tina [email protected] 65 Kibler, Clayton [email protected] 133 Koehler, Jane [email protected] 75 Kokhreidze, M. [email protected] 121 Kolo, Agatha [email protected] 131 Larson, Charles [email protected] 132 Latomanski, Eleanor [email protected] 34 Lee, Jung Keun [email protected] 79 Levin, Michael [email protected] 78 Lin, Mingqun [email protected] 156 Lina, Taslima T. [email protected] 70 Linsuwanon, Piyada [email protected] 114 Liu, Jiayou [email protected] 89 Luce-Fedrow, Alison [email protected] 116 Lugo-Caballero, Cesar [email protected] 19, 20, 125 Luo, Tian [email protected] 41 Lynn, Geoff [email protected] 38 Macaluso, Kevin R. [email protected] 1 Maina, Alice [email protected] 4 Mamisashvili, Eliso [email protected] 15 Martinez, Juan [email protected] 139 McBride, Jere W. [email protected] 66 McGill, Jodi [email protected] 82 Miller, Heather [email protected] 104 Minnick, Michael [email protected] 53 Mitra, Shubhajit [email protected] 137 Moore, James D. [email protected] 107 Moormeier, Derek [email protected] 51 Mullins, Kristin [email protected] 154 Narra, Hema [email protected] 103 Neelakanta, Girish [email protected] 74 Nicholson, William [email protected] 91 Noh, Susan [email protected] 100 Noriea, Nicholas [email protected] 64, 143 O’Conor, Kate [email protected] 155 Oki, Aminat [email protected] 55 Oliver, Jonathan [email protected] 39 Osterbind, Audrey [email protected] 7 Paddock, Chris [email protected] 3 Paris, Daniel [email protected] 80 Patel, Jignesh [email protected] 29 106 Paulauskas, Algimantas [email protected] 92 Pedra, Joao [email protected] 73 Perniciaro, Jamie [email protected] 128 Priestley, Rachael [email protected] 50 Raghavan, Rahul [email protected] 13 Raghavan, Ram [email protected] 90 Reif, Kathryn [email protected] 105 Richards, Allen [email protected] 5 Rikihisa, Yasuko [email protected] Plenary Session, 56 Riley, Sean [email protected] 33 Rodino, Kyle [email protected] 42 Rodkvamtook, Wuttikon [email protected] 26 Sahni, Abha [email protected] 59 Samuel, James E. [email protected] 8 Sandoz, Kelsi [email protected] 11 Santanello, Catherine [email protected] 110 Schafer, Walter [email protected] 106 Schroeder, Casey [email protected] 101 Shaw, Dana [email protected] 37 Shaw, Edward [email protected] 47 Shelite, Thomas [email protected] 138 Shin, Sunny [email protected] 147 Simoes, Isaura [email protected] 144 Smalley, Claire [email protected] 60 Soong, Lynn [email protected] 83 St. John, Heidi [email protected] 95 Stenos, John [email protected] 115 Stokes, John [email protected] 30 Stromdahl, Ellen [email protected] 94 Sunyakumthorn, Piyanate [email protected] 48, 117 Teague, Kristine [email protected] 31 Trounson, Vaughan [email protected] 36 Turco, Jenifer [email protected] 62 Utennam, Darunee [email protected] 111 Varela-Stokes, Andrea [email protected] 2 Velayutham, Thangam Sudha [email protected] 46 Wachter, Shaun [email protected] 99 Walker, David [email protected] 146 Wang, Ying [email protected] 71 Wang, Xiaowei [email protected] 58 Wang, Jennifer [email protected] 135 Weber, Mary [email protected] 140 Wei, Lanjing [email protected] 72 Welch, Matthew [email protected] 67 Winchell, Caylin [email protected] 10 Xu, Guang [email protected] 148 Zhgenti, Ekaterine [email protected] 93 Zhong, Jianmin [email protected] 76 Zhu, Bing [email protected] 108

107 28th Meeting of the American Society for Rickettsiology 2015-2017 ASR Executive Council President: Jason Carlyon, PhD Vice President: J. Stephen Dumler, MD Department of Microbiology and Joint Department of Pathology Immunology Uniformed Services University of the Health School of Medicine Sciences Virginia Commonwealth University Walter Reed National Military Medical Center

Secretary-Treasurer: Kevin R. Macaluso, PhD Councilor-at-Large: Daniel Voth, PhD Pathobiological Sciences Department of Microbiology and School of Veterinary Medicine Immunology Louisiana State University University of Arkansas for Medical Sciencies

Past President: Jere W. McBride, PhD Department of Pathology University of Texas Medical Branch

2016 Scientific Committee All Executive Council Members, plus the following ASR members: Chris Paddock, MD Roman Ganta, PhD Ulrike Munderloh, PhD Centers for Disease Control Department of Diagnostic Department of Entomology and Prevention Medicine/Pathobiology University of Minnesota Rickettsial Zoonoses Branch Kansas State University

Joao Pedra, PhD Robert Heinzen, PhD Department of Microbiology and Laboratory of Bacteriology Immunology Rocky Mountain Laboratories School of Medicine NIAID/National Institutes of Health University of Maryland

2016 Nominations Committee Chris Paddock, MD Wendy Brown, MPH, PhD Centers for Disease Control Department of Veterinary Microbiology and and Prevention Pathology Rickettsial Zoonoses Branch Washington State University

Ulrike Munderloh, PhD David Walker, MD Department of Entomology Department of Pathology University of Minnesota University of Texas Medical Branch

Robert Heinzen, PhD Laboratory of Bacteriology Rocky Mountain Laboratories NIAID/National Institutes of Health

108 Workshop Organizing Committee (* denotes member of the ASR Executive Council):

Jason Carlyon* – Virginia Commonwealth University J. Stephen Dumler* – Uniformed Services University of the Health Sciences Kevin Macaluso* – Louisiana State University Daniel Voth* – University of Arkansas for Medical Sciences Jere McBride* – University of Texas Medical Branch Ulrike Munderloh – University of Minnesota Nahed Ismail – University of Pittsburg Guy Palmer – Washington State University Juan Martinez – Louisiana State University David Walker – University of Texas Medical Branch Jim Samuel – Texas A & M University Susan Noh – Washington State University Wendy Brown – Washington State University

Workshop Scientific Committee

Yasuko Rikihisa – Ohio State University Stacey Gilk – University of Arkansas for Medical Sciences Joao Pedra – University of Maryland Roman Ganta – Kansas State University Michael Levin – Centers for Disease Control Abdu Azad – University of Maryland Bob Heinzen – Rocky Mountain Laboratories, NIAID Chris Paddock – Centers for Disease Control Daniel Paris – Mahidol-Oxford Research Unit, Bangkok, Thailand Girish Neelakanta – Old Dominion University Janet Foley – University of California Davis

ASR Meeting Coordinator

Josh Hilbrand Global Campus Kansas State University 1615 Anderson Avenue Manhattan, KS 66502 [email protected] 785-532-2495

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